RESUMEN
This composite article is intended to give the experts in the field of cochlear mechanics an opportunity to voice their personal opinion on the one mechanism they believe dominates cochlear amplification in mammals. A collection of these ideas are presented here for the auditory community and others interested in the cochlear amplifier. Each expert has given their own personal view on the topic and at the end of their commentary they have suggested several experiments that would be required for the decisive mechanism underlying the cochlear amplifier. These experiments are presently lacking but if successfully performed would have an enormous impact on our understanding of the cochlear amplifier.
Asunto(s)
Cóclea/fisiología , Audición , Mecanotransducción Celular , Animales , Percepción Auditiva , Movimiento Celular , Retroalimentación Fisiológica , Células Ciliadas Auditivas/fisiología , Humanos , Transporte Iónico , Potenciales de la Membrana , Modelos Biológicos , Presión , Sonido , VibraciónRESUMEN
Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.
Asunto(s)
Modelos Animales de Enfermedad , Células Ciliadas Auditivas Externas/fisiología , Proteínas Motoras Moleculares/genética , Animales , Umbral Auditivo/fisiología , Potenciales Microfónicos de la Cóclea/genética , Exones/genética , Marcación de Gen , Genotipo , Células Ciliadas Auditivas Externas/patología , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Microscopía Confocal , Fenotipo , ARN Mensajero/genéticaRESUMEN
Targeted deletion of the prestin gene reduces cochlear sensitivity and eliminates both frequency selectivity and outer hair cell (OHC) somatic electromotility. In addition, it has been reported by Liberman and colleagues that F2 generation heterozygotes exhibit a 6 dB reduction in sensitivity, as well as a decrease in protein and electromotility. Considering that the active process is non-linear, a halving of somatic electromotility would be expected to produce a much larger change in sensitivity. We therefore re-evaluated comparisons between heterozygotes and wildtype mice using both in vivo and in vitro electrophysiology, as well as molecular biology. Data reported here for F3-F5 generation mice indicate that compound action potential thresholds and tuning curves, as well as the cochlear microphonic, are similar in heterozygotes and wildtype controls. Measurements of non-linear capacitance in isolated OHCs demonstrate that charge density, as well as the voltage dependence and sensitivity of motor function, is indistinguishable in the two genotypes, as is somatic electromotility. In addition, both immunocytochemistry and western blot analysis in young adult mice suggest that prestin protein in heterozygotes is near normal. In contrast, prestin mRNA is always less than in wildtype mice at all ages tested. Results from F3-F5 generation mice suggest that one copy of the prestin gene is capable of compensating for the deleted copy and that heterozygous mice do not suffer peripheral hearing impairment.
Asunto(s)
Percepción Auditiva/fisiología , Cóclea/fisiología , Dosificación de Gen/fisiología , Proteínas/fisiología , Animales , Eliminación de Gen , Ratones , Ratones Noqueados , Proteínas Motoras MolecularesRESUMEN
Gross-potential recordings in mice lacking the Prestin gene indicate that compound action potential (CAP) thresholds are shifted by approximately 45 dB at 5 kHz and by approximately 60 dB at 33 kHz. However, in order to conclude that outer hair cell (OHC) electromotility is associated with the cochlear amplifier, frequency selectivity must be evaluated and the integrity of the OHC's forward transducer ascertained. The present report demonstrates no frequency selectivity in CAP tuning curves recorded in homozygotes. In addition, CAP input-output functions indicate that responses in knockout mice approach those in controls at high levels where the amplifier has little influence. Although the cochlear microphonic in knockout mice remains approximately 12 dB below that in wild-type mice even at the highest levels, this deficit is thought to reflect hair cell losses in mice lacking prestin. A change in OHC forward transduction is not implied because knockout mice display non-linear responses similar to those in controls. For example, homozygotes exhibit a bipolar summating potential (SP) with positive responses at high frequencies; negative responses at low frequencies. Measurement of intermodulation distortion also shows that the cubic difference tone, 2f(1)-f(2), is approximately 20 dB down from the primaries in both homozygotes and their controls. Because OHCs are the sole generators of the negative SP and because 2f(1)-f(2) is also thought to originate in OHC transduction, these data support the idea that forward transduction is not degraded in OHCs lacking prestin. Finally, application of AM1-43, which initially enters hair cells through their transducer channels, produces fluorescence in wild-type and knockout mice indicating transducer channel activity in both inner and outer hair cells.
Asunto(s)
Estimulación Acústica , Potenciales de Acción/genética , Cóclea/fisiología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas/genética , Estimulación Acústica/métodos , Potenciales de Acción/fisiología , Animales , Cóclea/metabolismo , Colorantes Fluorescentes/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Motoras Moleculares , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas/metabolismoRESUMEN
The influence of increased intracellular calcium level on outer hair cell (OHC) electromotility was examined by means of transcellular electrical stimulation in a partitioning microchamber. Electromotile activity was measured before and after application of the calcium ionophore ionomycin, which promotes the inflow of extracellular calcium, as well as its release from intracellular calcium stores. The ionomycin solvent, dimethyl sulphoxide (DMSO), by itself elicited a significant decrease in the magnitude of OHC electromotility. The DMSO effect was counteracted by 10 microM ionomycin and was reversed by 50 microM ionomycin. The increase in electromotility is partially mediated by a calmodulin-dependent mechanism, since W7, a calmodulin antagonist, attenuated the 50 microM ionomycin-induced motility increase. Our results suggest that the electromotility magnitude increase in isolated OHCs due to ionomycin is a calcium/calmodulin-dependent phenomenon.
Asunto(s)
Calcio/fisiología , Células Ciliadas Auditivas Externas/fisiología , Animales , Calmodulina/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Técnicas In Vitro , Ionomicina/farmacología , Ionóforos/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Efferents, originating in the superior olivary complex, preferentially synapse with cochlear outer hair cells (OHCs), with acetylcholine (ACh) as their primary neurotransmitter. The OHC ACh receptors (AChRs), which have unusual pharmacology, have been cloned and identified as a new subunit (alpha9) of the nicotinic AChR family. The expression of alpha9 AChRs is first detected before birth and peaks between 6 and 10 days after birth (DAB) in developing mice and rats, while functional maturation of the receptor, as determined by measuring the ACh-induced currents, takes place between 6 and 12 DAB. In this study we attempted to examine the development of AChRs in OHCs grown in explanted cultures, deprived of efferent innervation. ACh-induced currents were used as an assay. Reverse transcription-PCR analysis was also performed to detect the expression of alpha9 subunit from cultured OHCs. PCR study indicates that mRNA of the alpha9 subunit was expressed in primary cochlear cultures, similar to that seen in the cochleae of developing animals. Measurement of whole-cell currents showed that ACh-induced outward current was first detected around 5 days in a fraction of cultured OHCs. The number of responsive cells increased between 5 and 12 days in culture. The size of ACh-induced currents also increased during this period. These results suggest that the development of AChRs in cultured OHCs is not affected by removal of efferent innervation.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Receptores Colinérgicos/metabolismo , Acetilcolina/farmacología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Membrana Basilar/crecimiento & desarrollo , Técnicas de Cultivo , Conductividad Eléctrica , Gerbillinae , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/fisiología , Órgano Espiral/crecimiento & desarrollo , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Inner hair cell (IHC) responses to tone-burst stimuli were measured from three locations in the apical half of the guinea pig cochlea. In addition to the measurement of ac receptor potentials, average intracellular voltages, reflecting both ac and dc components of the receptor potential, were computed and compared to determine how bandwidth changes with level. Companion phase measures were also obtained and evaluated. Data collected from turn 2, where best frequency (BF) is approximately 4000 Hz, indicate that frequency response functions are asymmetrical with steeper slopes above the best frequency of the cell. However, in turn 4, where BF is around 250 Hz, the opposite behavior is observed and the steepest slopes are measured below BF. The data imply that cochlear filters are generally asymmetrical with steeper slopes above BF. High-pass filtering by the middle ear serves to reduce this asymmetry in turn 3 and to reverse it in turn 4. Apical response patterns are used to assess the degree to which the middle ear transfer function, the IHC's velocity dependence and the shunting effect of the helicotrema influence low-frequency hearing in guinea pigs. Implications for low-frequency hearing in man are also discussed.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Audición/fisiología , Percepción de la Altura Tonal/fisiología , Animales , Umbral Auditivo/fisiología , Cóclea/fisiología , Potenciales Evocados Auditivos/fisiología , Cobayas , Humanos , Espectrografía del Sonido , Especificidad de la EspecieRESUMEN
Computer modeling of the outer hair cell (OHC) motor protein prestin produces ambiguous results regarding transmembrane regions and localization of its termini. To determine the location of prestin's N- and C-termini, we created prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was studied in prestin-transfected cells using immunofluorescence. In permeabilized cells, antibodies label the plasma membrane of 30% of the cells, reflecting transfec- tion efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These data suggest that prestin's N-and C-termini are cytoplasmic. Furthermore, prestin staining in OHCs was observed only under permeabilizing conditions. These results implicate prestin's N- and C-termini as portions that may interact with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results.
Asunto(s)
Membrana Celular/química , Epítopos/química , Células Ciliadas Auditivas Externas/química , Proteínas/química , Animales , Especificidad de Anticuerpos/genética , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , ADN Complementario/genética , Epítopos/genética , Técnica del Anticuerpo Fluorescente , Gerbillinae , Proteínas Fluorescentes Verdes , Células Ciliadas Auditivas Externas/ultraestructura , Audición/fisiología , Indicadores y Reactivos/análisis , Proteínas Luminiscentes/análisis , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Biológicos , Estructura Terciaria de Proteína/fisiología , Conejos , Transducción de Señal/fisiología , TransfecciónRESUMEN
Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.
Asunto(s)
Bicarbonatos/metabolismo , Cloruros/metabolismo , Células Ciliadas Auditivas Externas/fisiología , Proteínas/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de Transporte de Anión , Aniones/farmacología , Bicarbonatos/farmacología , Células CHO , Cationes/farmacología , Membrana Celular/metabolismo , Cloruros/farmacología , Cricetinae , Conductividad Eléctrica , Electrofisiología , Modelos Biológicos , Mutación , Técnicas de Placa-Clamp , Conformación Proteica , Proteínas/química , Proteínas/genética , Ratas , Transportadores de SulfatoRESUMEN
Isolated guinea-pig outer hair cells (OHCs) (n = 52) were inserted into a partitioning microchamber and electromotility was measured by a calibrated optoelectronic apparatus. Acetylcholine (ACh), and ACh together with different protein kinase inhibitors, were applied to OHCs through a puffer pipette. ACh produced a magnitude increase of electromotility. This magnitude increase was inhibited by co-application of KN-62, a calcium/calmodulin-dependent protein kinase II (CAMKII) inhibitor. Simultaneous application of ACh and H-89, a selective protein kinase A (PKA) inhibitor, did not antagonize the ACh response. Further support for the CAMKII-mediated ACh influence on electromotility is that the magnitude increase is also inhibited by the calmodulin antagonist trifluoperazine (TFP) and by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. The results suggest an essential role of calcium in the ACh-mediated increase of the magnitude of electromotility. Elevation of the intracellular calcium concentration apparently activates CAMKII which, in turn, phosphorylates membrane or cytoskeletal substrate(s). This molecular modification probably leads to reduced axial cell stiffness and subsequent increase of the electromotile response.
Asunto(s)
Acetilcolina/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Movimiento Celular/fisiología , Células Ciliadas Auditivas Externas/fisiología , Transmisión Sináptica/fisiología , Adenosina Trifosfatasas/fisiología , Animales , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Células Cultivadas , Cobayas , Potenciales de la Membrana/fisiología , FosforilaciónRESUMEN
1. Under whole-cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage-dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane-bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage-driven mechanical activity, which spans the audio spectrum. 2. Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (Q(max) = 0.99 +/- 0.16 pC, z = 0.88 +/- 0.02; n = 5, means +/- S.E.M.). Unlike that of the adult OHC, the voltage at peak capacitance (V(pkcm)) was very negative (-74.7 +/- 3.8 mV). Linear capacitance in transfected cells was 43.7 +/- 13.8 pF and membrane resistance was 458 +/- 123 Mohms. 3. Voltage steps from the holding potential preceding the measurement of capacitance-voltage functions caused a time- and voltage-dependent shift in V(pkcm). For a prepulse to -150 mV, from a holding potential of 0 mV, V(pkcm) shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (tau(0) = 51.6 ms, tau(1) = 8.5 s) similar to that of the native gerbil OHC. 4. Membrane tension, delivered by increasing pipette pressure, caused a positive shift in V(pkcm). A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5. Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestin's activity.
Asunto(s)
Células Ciliadas Auditivas Externas/fisiología , Proteínas/fisiología , Animales , Proteínas de Transporte de Anión , Línea Celular , Membrana Celular/metabolismo , Conductividad Eléctrica , Impedancia Eléctrica , Gerbillinae , Células Ciliadas Auditivas Externas/metabolismo , Humanos , Activación del Canal Iónico , Potenciales de la Membrana/fisiología , Conformación Molecular , Técnicas de Placa-Clamp , Proteínas/química , Estrés Mecánico , Transportadores de Sulfato , Factores de TiempoRESUMEN
In order to identify hair cell specific genes, it is essential to obtain isolated hair cells in quantity. While whole-cell recordings have been made from isolated inner hair cells (IHCs) from guinea pigs, detailed methods for obtaining a fairly large amount of isolated inner hair cells have not been published. Here we describe a protocol that can yield a fairly large amount of isolated gerbil IHCs. This technique can provide sufficient numbers of solitary IHCs for either electrophysiological studies of the cell's membrane properties or identifying genes related to IHC functions using techniques of molecular biology.
Asunto(s)
Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Animales , Movimiento Celular/fisiología , Separación Celular/métodos , Estimulación Eléctrica , Gerbillinae , Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/fisiología , Técnicas de Placa-ClampRESUMEN
The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a 'gating current', which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.
Asunto(s)
Células Ciliadas Auditivas Externas/fisiología , Proteínas de Transporte de Membrana , Proteínas Motoras Moleculares , Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Anión , Fenómenos Biomecánicos , Proteínas Portadoras/fisiología , Línea Celular , Movimiento Celular , Clonación Molecular , ADN Complementario , Conductividad Eléctrica , Expresión Génica , Gerbillinae , Células Ciliadas Auditivas Externas/química , Humanos , Proteínas Motoras Moleculares/efectos de los fármacos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas/análisis , Proteínas/genética , Homología de Secuencia de Aminoácido , Salicilato de Sodio/farmacología , Transportadores de Sulfato , Distribución Tisular , TransfecciónRESUMEN
Inner hair cell (IHC) and organ of Corti (OC) responses are measured from the apical three turns of the guinea pig cochlea, allowing access to regions with best, or most sensitive, frequencies at approximately 250, 1000, and 4000 Hz. In addition to measuring both ac and dc receptor potentials, the average value of the half-wave rectified response (AVEHR) is computed to better reflect the signal that induces transmitter release. This measure facilitates comparisons with single-unit responses in the auditory nerve. Although IHC ac responses exhibit compressive growth, response magnitudes at high levels depend on stimulus frequency. For example, IHCs with moderate and high best frequencies (BF) exhibit more linear responses below the BF of the cell, where higher sound-pressure levels are required to approach saturation. Because a similar frequency dependence is observed in extracellular OC responses, this phenomenon may originate in cochlear mechanics. At the most apical recording location, however, the pattern documented at the base of the cochlea is not seen in IHCs with low BFs around 250 Hz. In fact, more linear behavior is measured above the BF of the cell. These frequency-dependent features require modification of cochlear models that do not provide for longitudinal variations and generally depend on a single stage of saturation located at the synapse. Finally, behavior of dc and AVEHR responses suggests that a single IHC is capable of coding intensity over a large dynamic range [Patuzzi and Sellick, J. Acoust. Soc. Am. 74, 1734-1741 (1983); Smith et al., in Hearing--Physiological Bases and Psychophysics (Springer, Berlin, 1983); Smith, in Auditory Function (Wiley, New York, 1988)] and that information compiled over wide areas along the cochlear partition is not essential for loudness perception, consistent with psychophysical results [Viemeister, Hearing Res. 34, 267-274 (1988)].
Asunto(s)
Órgano Espiral/fisiología , Animales , Fenómenos Biomecánicos , Cóclea/fisiología , Cobayas , Células Ciliadas Auditivas Internas/fisiología , TransductoresRESUMEN
A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.
Asunto(s)
Cóclea/crecimiento & desarrollo , Oído Interno/crecimiento & desarrollo , Gerbillinae/crecimiento & desarrollo , Animales , Membrana Basilar/crecimiento & desarrollo , Órgano Espiral/citología , Órgano Espiral/crecimiento & desarrollo , Membrana Tectoria/crecimiento & desarrolloRESUMEN
Cochlear outer hair cells change their axial dimension and theiraxial stiffness when their membrane potential is altered. These changes appear to be highly correlated. Because of this, we endeavored to produce models that would yield both phenomena via a single mechanism. Two models are proposed. In one, it is assumed that elementary motor molecules can be in either of two conformational states, these having different physical lengths and stiffnesses. The state of the molecule is taken to be a stochastic function of membrane potential and is expressed by a Boltzmann relationship. In the other model, a similar dependence is assumed to occur between membrane potential and stiffness, but no dimensional change isassigned to the molecule. Length changes can be had by preloading the cell. We show that either general model can produce realistic length and stiffness changes with an appropriate selection of parameters. One particular realization of the first model is proposed as an example. In this--the boomerang model--the molecule is assumed to be L-shaped, with two different angles between the two legs representing the conformational states. Finally, the behavior of the model is compared with available data when the voltage stimulus comprises a brief sinusoid upon a DC pedestal.
Asunto(s)
Células Ciliadas Auditivas Externas/fisiología , Modelos Neurológicos , Animales , Movimiento Celular , Elasticidad , HumanosRESUMEN
We have shown recently that isolated cochlear outer hair cells change their axial stiffness when their membrane potential is altered under voltage-clamp. Here we extend those observations, using a more stable mechanical platform, the microchamber, to hold the cells and to deliver voltage commands. Cell stiffness is determined by opto-electronically measuring the amplitude of motion of a flexible fiber as it is loaded by the cell. Cell stiffness is decreased by depolarization and increased by hyperpolarization. The stiffness changes have been measured with sinusoidal electrical command signals up to 1750 Hz and fiber motion up to 2000 Hz. It is shown that electrically evoked stiffness changes and length changes (electromotility) have very similar characteristics and may arise in a common process.
Asunto(s)
Células Ciliadas Auditivas Externas/fisiología , Animales , Elasticidad , Electrofisiología , Gadolinio/farmacología , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Modelos Neurológicos , Movimiento (Física)RESUMEN
The aim of this study is to examine the effect of phosphorylation pathways on the electrically evoked fast motile response of isolated outer hair cells (OHCs). Transcellular electrical stimulation was applied in the microchamber to guinea pig OHCs and motility was measured before and after drug application. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) and dibutyryl 3',5'-cyclic guanosine monophosphate (cGMP agonist) were studied. As controls, L15 medium and dimethyl-sulfoxide (DMSO) were used. In each group, 12 cells were measured. Forskolin and PMA were dissolved in 0.1% DMSO to render them membrane permeable. DMSO by itself caused a statistically significant electromotility magnitude decrease. Forskolin and PMA could not reverse the motility decrease due to DMSO, the effects seen in their presence were the same as observed with DMSO alone. Thus, neither 3',5'-cyclic AMP-dependent protein kinase nor calcium/phospholipid-dependent protein kinase appear to have modulatory effects on electromotility. Dibutyryl cGMP (DBcGMP), in concentrations of 200 microM, elicited a significant electromotility magnitude increase. The DBcGMP effect could be inhibited by co-application of 200 microM DBcGMP and 100 microM 8-Rp-pCPT-cGMPS (8-4-chlorophenylthio-guanosine 3',5'-cyclic monophosphothioate, Rp isomer, a cGMP antagonist). Our results suggest that OHC electromotility is modulated by a cGMP-dependent pathway.
Asunto(s)
Movimiento Celular/fisiología , GMP Cíclico/metabolismo , Células Ciliadas Auditivas Externas/fisiología , Adenilil Ciclasas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Colforsina/farmacología , GMP Cíclico/agonistas , GMP Cíclico/antagonistas & inhibidores , GMP Dibutiril Cíclico/farmacología , Estimulación Eléctrica , Activación Enzimática/efectos de los fármacos , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/metabolismo , Técnicas In Vitro , Fosforilación , Proteína Quinasa C/metabolismo , Sistemas de Mensajero Secundario , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The basilar membrane in the mammalian cochlea vibrates when the cochlea receives a sound stimulus. This mechanical vibration is transduced into hair cell receptor potentials and thereafter encoded by action potentials in the auditory nerve. Knowledge of the mechanical transformation that converts basilar membrane vibration into hair cell stimulation has been limited, until recently, to hypothetical geometric models. Experimental observations are largely lacking to prove or disprove the validity of these models. We have developed a hemicochlea preparation to visualize the kinematics of the cochlear micromechanism. Direct mechanical drive of 1-2 Hz sinusoidal command was applied to the basilar membrane. Vibration patterns of the basilar membrane, inner and outer hair cells, supporting cells, and tectorial membrane have been recorded concurrently by means of a video optical flow technique. Basilar membrane vibration was driven in a direction transversal to its plane. However, the direction of the resulting vibration was found to be essentially radial at the level of the reticular lamina and cuticular plates of inner and outer hair cells. The tectorial membrane vibration was mainly transversal. The transmission ratio between cilia displacement of inner and outer hair cells and basilar membrane vibration is in the range of 0.7-1.1. These observations support, in part, the classical geometric models at low frequencies. However, there appears to be less tectorial membrane motion than predicted, and it is largely in the transversal direction.