Does BAPTA leave outer hair cell transduction channels closed?

The calcium chelator BAPTA was iontophoresed into the scala media of the second turn of the guinea pig cochlea. This produced a reduction in low frequency cochlear microphonic (CM) measured in scala media and an elevation of the cochlear action potential (CAP) threshold that lasted for the duration of the experiment. Using two pipettes, one filled with KCl and the other KCl and BAPTA (50, 20 and 5 mM) it was possible to observe the effect of passing current through one electrode while measuring the endolymphatic potential (EP) with the other. The results demonstrated that current passed via the BAPTA pipette caused a sustained increase in EP of 8.2, 12.9 and 7.8 mV in the three animals used. This increase coincided with the decrease in low frequency CM that indicated a causal connection between the two. In a second series of experiments, pipettes with larger tips were inserted into scala media in the first cochlear turn and BAPTA was allowed to diffuse from the pipette. The results confirmed the relationship between EP increase and the fall of scala media CM. One interpretation of these results is that lowering the Ca2+ concentration of endolymph with BAPTA inhibits mechano-electrical transduction in outer hair cells (OHCs) and leaves the hair cell transduction channels in a closed state, thus increasing the resistance across OHCs and increasing the EP. These findings are consistent with a model of hair cell transduction in which tension on stereo cilia opens the transduction channels.

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis.

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.

Effects of 4-aminopyridine on electrically evoked cochlear emissions and mechano-transduction in guinea pig outer hair cells.

Stimulation of the cochlea with alternating current produces sound in the ear canal. These electrically evoked oto-acoustic emissions (EEOAEs) are attributed to electro-motility of outer hair cells (OHCs). Earlier work suggested EEOAEs were sensitive to the open probability of OHC mechano-electrical transduction (MET) channels. They were attenuated by 4-aminopyridine (4-AP) and amplitude-modulated by low frequency sound, consistent with current gaining access to a motility source via the MET conductance. However, inconsistencies in the behaviour as well as physical considerations argued against this simple interpretation. In this study the behaviour of EEOAEs in the presence of 4-AP in scala media was examined along with OHC transfer functions derived from low frequency cochlear microphonic (CM) waveforms. Both the level and the modulation of the EEOAEs were reduced by 4-AP, but disproportionately more so than the 4-AP-induced loss of CM. In addition, the modulation as well as the level of the EEOAEs recovered more rapidly than the CM. Both these results indicated that 4-AP modified the process of EEOAE generation independently of its effect on the gross receptor current through the MET conductance. Changes in the derived OHC transfer functions, specifically shifts in the estimated operating bias of the MET channels, indicated the effects of 4-AP applied to the endolymphatic surface of OHCs were complex. It is suggested that both direct and indirect consequences of a 4-AP blockade may have contributed. 4-AP was ineffective when applied to scala tympani.

Two enhancers and one silencer located in the introns of regA control somatic cell differentiation in Volvox carteri.

The regA gene plays a central role in germ-soma differentiation of Volvox carteri by suppressing all reproductive functions in somatic cells. Here we show that the minimal promoter of regA consists of only 42 bp immediately upstream of the transcription start site, and that it contains no discernible regulatory elements. However, introns 3 and 5 are both required for regA expression in somatic cells, and intron 7 is essential for silencing regA in gonidia (asexual reproductive cells). A regA gene lacking intron 7 rescues the normal phenotype of mutant somatic cells, but also results in gonidia that reproduce only weakly and soon die out. The same phenotype is observed when a regA gene containing intron 7 is placed under control of a constitutive promoter, suggesting that the silencing activity of intron 7 is promoter specific. Intron 7 is unusual in that it contains a potential ORF that is in frame with exons 7 and 8, and some transcripts are produced in which intron 7 is retained. However, a regulatory role for the intron 7 translation product can be ruled out, because a construct in which intron 7 must be translated, and one in which it cannot be translated, both result in wild-type development of both cell types. Furthermore, intron 7 is unable to act in trans to silence regA, but is able to exert its normal effect when placed in a different location within the gene. Therefore, it appears that intron 7 functions in gonidia as a classical cell-type-specific and promoter-specific enhancer, of the inhibitory type that is often referred to as a silencer.

In vivo evidence for a cochlear amplifier in the hair-cell bundle of lizards.

Vertebrate sensory hair cells achieve high sensitivity and frequency selectivity by adding self-generated mechanical energy to low-level signals. This allows them to detect signals that are smaller than thermal molecular motion and to achieve significant resonance amplitudes and frequency selectivity despite the viscosity of the surrounding fluid. In nonmammals, a great deal of in vitro evidence indicates that the active process responsible for this amplification is intimately associated with the hair cells' transduction channels in the stereovillar bundle. Here, we provide in vivo evidence of hair-cell bundle involvement in active processes. Electrical stimulation of the inner ear of a lizard at frequencies typical for this hearing organ induced low-level otoacoustic emissions that could be modulated by low-frequency sound. The unique modulation pattern permitted the tracing of the active process involved to the stereovillar bundles of the sensory hair cells. This supports the notion that, in nonmammals, the cochlear amplifier in the hair cells is driven by a bundle motor system.

Germ-soma differentiation in volvox.

Volvox carteri is a spherical green alga with a predominantly asexual mode of reproduction and a complete germ-soma division of labor. Its somatic cells are specialized for motility, incapable of dividing, and programmed to die when only a few days old, whereas its gonidia (asexual reproductive cells) are nonmotile, specialized for growth and reproduction, and potentially immortal. When a gonidium is less than 2 days old it divides to produce a juvenile spheroid containing all of the somatic cells and gonidia that will be present in an adult of the next generation. The first visible step in germ-soma differentiation is a set of asymmetric cleavage divisions in the embryo that set apart small somatic initials from their large gonidial-initial sister cells. Three types of genes have been found to play key roles in germ-soma specification. First a set of gls genes act in the embryos to shift cell-division planes, resulting in the asymmetric divisions that set apart the large-small sister-cell pairs. Then a set of lag genes act in the large cells to prevent somatic differentiation, while the regA gene acts in the small cells to prevent reproductive development. An inducible transposon was used to tag and recover some of these and other developmentally important genes. The glsA gene encodes a chaperone-like protein that, like another chaperone that is one of its putative binding partners, is associated with the cell division apparatus, although how this leads to asymmetric division remains to be elucidated. The regA gene encodes a somatic-cell-specific nuclear protein that appears to function by repressing genes required for chloroplast biogenesis, thereby preventing somatic cells from growing enough to reproduce. Somatic-cell-specific expression of regA is controlled by three intronic enhancers.

The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox.

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many region-specific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the 'flagellar hillocks' that encircle the basal ends of all flagella, and in the adjacent 'boundary zone' that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.

Changes to low-frequency components of the TEOAE following acoustic trauma to the base of the cochlea.

Several studies have shown that acoustic trauma to the base of the cochlea can result in loss of transient-evoked otoacoustic emission (TEOAE) energy at frequencies much lower than those affected in the audiogram. We have extended these studies to show that the low-frequency emission energy was substantially affected if the transient stimulus included frequencies within the range affected by the trauma, otherwise the change observed was small. In keeping with the suggestion that TEOAEs are predominantly comprised of intermodulation distortion energy (Yates and Withnell, Hear. Res. 136 (1999) 49-64), trauma to the basal region of the cochlea was found to affect emission energy across a broad frequency range in response to a wide-band acoustic stimulus. Further, group delay measurements demonstrated that the dominant contribution to the TEOAE originated from the basal region of the cochlea.

Evolution of multicellularity in the volvocine algae.

Recent studies reveal that relationships among the volvocine algae are more complex than was previously believed. Nevertheless, this group still appears to provide an unrivaled opportunity to analyze an evolutionary pathway leading from unicellularity (Chlamydomonas) to multicellularity with division of labor (Volvox). Significant progress in this regard was made in the past year when two genes playing key roles in Volvox cellular differentiation were cloned, and clues were uncovered regarding their mechanisms of action.

Prevalence of chief complaints in a pediatric clinic population.

PURPOSE: To determine the type and frequency of chief complaints reported by parents of pediatric patients younger than 8 years of age in an optometry clinic. There have been many reports on prevalence of pediatric eye and vision disorders; however, the frequency of presenting complaints has not been carefully investigated. METHODS: 578 patient charts were retrospectively evaluated to determine the entering chief complaint, representing a 28-month time period between January 1993 and April 1995. The subjects were classified by age, and the chief complaint was placed in one of eight categories. RESULTS: The most frequently reported (30.1%) entering complaint for all subjects was the need for a routine vision examination. Each age category, however, had a unique distribution of complaints. Infants (1 to 11 months of age, n = 24) and young school age children (6 to 7-11 years of age, N = 233) most frequently reported complaints in the "other" category, at rates of 37.5% and 38.2%, respectively. Toddlers (12 to 36 months of age, N = 61) most commonly presented with complaints of an observed eye turn (39.3%), and preschoolers (3 to 5-11 years of age, N = 260) presented mainly for routine vision examinations (48.9%). CONCLUSIONS: There was a large percentage of patients presenting for routine vision care, which may represent an encouraging trend in public education efforts concerning children's vision care needs. The relative frequency of chief complaints in each age category prepares the eye care practitioner to address common parental concerns. This information can help to improve vision care education and services for the pediatric population.

regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein.

Volvox has two cell types: mortal somatic cells and immortal germ cells. Here we describe the transposon-tagging, cloning and characterization of regA, which plays a central role as a master regulatory gene in Volvox germ-soma differentiation by suppressing reproductive activities in somatic cells. The 12.5 kb regA transcription unit generates a 6,725 nucleotide mRNA that appears at the beginning of somatic cell differentiation, and that encodes a 111 kDa RegA protein that localizes to the nucleus, and has an unusual abundance of alanine, glutamine and proline. This is a compositional feature shared by functional domains of many 'active' repressors. These findings are consistent with the hypothesis that RegA acts in somatic cells to repress transcription of genes required for growth and reproduction, including 13 genes whose products are required for chloroplast biogenesis.

glsA, a Volvox gene required for asymmetric division and germ cell specification, encodes a chaperone-like protein.

The gls genes of Volvox are required for the asymmetric divisions that set apart cells of the germ and somatic lineages during embryogenesis. Here we used transposon tagging to clone glsA, and then showed that it is expressed maximally in asymmetrically dividing embryos, and that it encodes a 748-amino acid protein with two potential protein-binding domains. Site-directed mutagenesis of one of these, the J domain (by which Hsp40-class chaperones bind to and activate specific Hsp70 partners) abolishes the capacity of glsA to rescue mutants. Based on this and other considerations, including the fact that the GlsA protein is associated with the mitotic spindle, we discuss how it might function, in conjunction with an Hsp70-type partner, to shift the division plane in asymmetrically dividing cells.

Volvox germline-specific genes that are putative targets of RegA repression encode chloroplast proteins.

In Volvox carteri, regA acts as a master gene to suppress all germ cell functions in somatic cells. Its product, RegA, has features of a transcriptional repressor. Here we report cDNA sequences representing 15 nuclear genes with properties expected of RegA targets: they are expressed strongly in germ cells and in regA-, but not regA+, somatic cells. Two of them encode polypeptides with no recognizable features, but ten (like three previously sequenced ones) encode chloroplast proteins of known function, and the remaining three encode putative chloroplast proteins of unknown function. This suggests that RegA blocks reproductive development in somatic cells by preventing chloroplast biogenesis, thereby making it impossible for the cells to grow enough to reproduce.

Enhancement of electrically evoked oto-acoustic emissions associated with low-frequency stimulus bias of the basilar membrane towards scala vestibuli.

Electrically evoked oto-acoustic emissions (EEOAEs) are sounds present in the ear canal when ac current is passed into the cochlea. EEOAEs are attributed to the activation of fast electromotile responses in outer hair cells (OHCs). An interesting property of EEOAEs is the phenomenon of "acoustic enhancement," where the emission amplitude is increased by moderate-level sound [D. C. Mountain and A. E. Hubbard, Hear. Res. 42, 195-202 (1989)]. In this report a form of enhancement is described which occurs with displacements of the basilar membrane toward scala vestibuli, during amplitude modulation of the EEOAE waveform by low-frequency tones. This "SV-bias enhancement" possibly consists of two components: (i) a low-level component induced by sound at levels which produce nonlinear growth of the cochlear microphonic and which may be equivalent to the "acoustic enhancement" described previously, and (ii) a high-level component which occurs at sound levels well above those which cause saturation of the cochlear microphonic. The low-level component could be explained by either an increased access of the extrinsically applied current to a membrane-based source of OHC motility, perhaps coupled with a reduction in negative feedback, or an increase in electromotile output during scala vestibuli displacements, but the origin of the high-level component is obscure.

ATP in endolymph enhances electrically-evoked oto-acoustic emissions from the guinea pig cochlea.

ATP was iontophoresed into the scala media of the guinea pig cochlea. A reversible increase in the amplitude of electrically-evoked oto-acoustic emissions (EEOAEs), and reductions in the endocochlear potential (EP) and the cochlear microphonic (CM) were observed. These effects were consistent with an action of ATP on P2X receptors on outer hair cells (OHC). The results confirm that endogenous endolymphatic ATP, acting via P2X receptors on OHCs, may serve a regulatory function in the cochlea.

Otoacoustic emissions measured with a physically open recording system.

Otoacoustic emissions have historically been measured with an acoustical probe assembly hermetically sealed in the ear canal, imposing in most cases a limited stimulus bandwidth. A physically open recording system should afford the possibility of a greater stimulus bandwidth but the change in acoustical load may affect the magnitude of otoacoustic emissions obtained. Here it is reported that the authors have measured in the guinea pig transient-evoked otoacoustic emissions extending in frequency to 20 kHz and cubic distortion tone otoacoustic emissions for f2 = 4737 and 8096 Hz with a physically open sound system. To address the effect of acoustical load provided by a physically open versus hermetically sealed system, the authors compared the amplitude of electrically evoked otoacoustic emissions recorded from a guinea pig in each case. The change in acoustical load in the ear canal introduced by the change in recording setup did not appear to make a substantial difference to the magnitude of otoacoustic emissions measured. A physically open recording system provides a good alternative to traditional acoustical probe assemblies sealed in the ear canal for the laboratory measurement of acoustically evoked otoacoustic emissions, with the advantage of permitting a greater stimulus bandwidth.

Cochlear electrically evoked emissions modulated by mechanical transduction channels.

Cochlear outer hair cells are capable of both mechanical-to-electrical and electrical-to-mechanical transduction. Vibration of their stereocilia by sound is believed to stimulate somatic motility via a receptor potential developed across the basolateral membrane, thereby enhancing the mechanical vibration and increasing the sensitivity and frequency selectivity of the ear. Extrinsic electrical currents, applied at the tops of the cells, also appear to activate motility in vivo, presumably after entering the cell. Earlier experiments suggested such currents might enter through the transduction channels themselves, but an alternative shunt pathway through the membrane capacitance seems more likely on physical grounds. We therefore recorded electrically evoked oto-acoustic emissions while modulating the transduction channels by driving them with low-frequency sound. Recordings of the low-frequency cochlear microphonic provided a measure of the mean electrical conductance through the channels during sound stimulation. Emissions increased during displacement of the basilar membrane toward scala vestibuli, when the channels were biased open, and decreased on the opposite phase, and the modulation of the emission was in direct proportion to the cochlear microphonic. The results are the strongest evidence yet that electrically evoked emissions are generated directly by mechanisms related to cochlear transduction and lead to the surprising conclusion that, for frequencies up to at least 12 kHz, extrinsic electrical currents enter the hair cell predominantly by the resistive pathway through the transduction channels. Alternatively, the results might be consistent with direct modulation of a motility source driven by capacitive currents but whose output depends on the state of the channels.