Biomedical subjects

J Santos-Sacchi

Publications and source records attributed to J Santos-Sacchi.

At least 37 records · Page 2Linked to original sources

PubMed1995

Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B.

Myosin VIIa is a newly identified member of the myosin superfamily of actin-based motors. Recently, the myosin VIIa gene was identified as the gene defective in shaker-1, a recessive deafness in mice [Gibson, F., Walsh, J., Mburu, P., Varela, A., Brown, K.A., Antonio, M., Beisel, K.W., Steel, K.P. & Brown, S.D.M. (1995) Nature (London) 374, 62-64], and in human Usher syndrome type 1B, an inherited disease characterized by congenital deafness, vestibular dysfunction, and retinitis pigmentosa [Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, F., Walsh, J., Mburu, P., Varela, A., Levilliers, J., Weston, M.D., Kelley, P.M., Kimberling, W.J., Wagenaar, M., Levi-Acobas, F., Larget-Piet, D., Munnich, A., Steel, K.P., Brown, S.D.M. & Petit, C. (1995) Nature (London) 374, 60-61]. To understand the normal function of myosin VIIa and how it could cause these disease phenotypes when defective, we generated antibodies specific to the tail portion of this unconventional myosin. We found that myosin VIIa was expressed in cochlea, retina, testis, lung, and kidney. In cochlea, myosin VIIa expression was restricted to the inner and outer hair cells, where it was found in the apical stereocilia as well as the cytoplasm. In the eye, myosin VIIa was expressed by the retinal pigmented epithelial cells, where it was enriched within the apical actin-rich domain of this cell type. The cell-specific localization of myosin VIIa suggests that the blindness and deafness associated with Usher syndrome is due to lack of proper myosin VIIa function within the cochlear hair cells and the retinal pigmented epithelial cells.

Abnormalities, Multiple↗

PubMed1995

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".

Animals↗

PubMed1994

Motility voltage sensor of the outer hair cell resides within the lateral plasma membrane.

The outer hair cell (OHC) from the organ of Corti is believed to be responsible for the mammal's exquisite sense of hearing. A membrane-based motile response of this cell underlies the initial processing of acoustic energy. The voltage-dependent capacitance of the OHC, possibly reflecting charge movement of the motility voltage sensor, was measured in cells during intracellular dialysis of trypsin under whole cell voltage clamp. Within 10 min after dialysis, light and electron microscopic examination revealed that the subplasmalemmal structures, including the cytoskeletal framework and subsurface cisternae, were disrupted and/or detached from adjacent plasma membrane. Dialysis of heat-inactivated trypsin produced no changes in cell structure. Simultaneous measures of linear and nonlinear membrane capacitance revealed minimal changes, indicating that contributions by subsurface structures to the generation of the nonlinear capacitance are unlikely. This study strongly suggests that voltage-dependent charge movement in the OHC reflects properties of the force generator's voltage sensor and that the sensor/motor resides solely within the lateral plasma membrane.

Animals↗

PubMed1994

Cell coupling in the supporting cells of Corti's organ: sensitivity to intracellular H+ and Ca+2.

The input capacitance of cell pairs or small groups can be used to gauge the degree of electrical coupling via gap junctions (Santos-Sacchi, 1991). In order to estimate junctional sensitivity to intracellular Ca+2 and H+ concentration, the input capacitance of supporting cell syncytia of the organ of Corti was measured with the whole cell voltage clamp technique, while directly modifying the cation concentrations via the patch pipette. Typically, a pH below 6.5 was capable of uncoupling Hensen cells. On the other hand, pCa levels as low as 3 were ineffective.

Animals↗

PubMed1993

Harmonics of outer hair cell motility.

The voltage-dependent mechanical activity of outer hair cells (OHC) from the organ of Corti is considered responsible for the peripheral auditory system's enhanced ability to detect and analyze sound. Nonlinear processes within the inner ear are presumed to be characteristic of this enhancement process. Harmonic distortion in the OHC mechanical response was analyzed under whole-cell voltage clamp. It is shown that the OHC produces DC, fundamental and second harmonic length changes in response to sinusoidal transmembrane voltage stimulation. Mechanical second harmonic distortion decreases with frequency, whereas the predicted transmembrane second harmonic voltage increases with frequency. Furthermore, the phase of the second harmonic distortion does not correspond to the phase of the predicted transmembrane voltage. In contradistinction, it has been previously shown (Santos-Sacchi, J. 1992. Neuroscience. 12:1906-1916) that fundamental voltage and evoked mechanical responses share magnitude and phase characteristics. OHC length changes are modeled as resulting from voltage-dependent cell surface area changes. The model suggests that the observed harmonic responses in the mechanical response are consistent with the nonlinearity of the voltage-to-length change (V-delta L) function. While these conclusions hold for the data obtained with the present voltage clamp protocol and help to understand the mechanism of OHC motility, modeling the electromechanical system of the OHC in the in vivo state indicates that the mechanical nonlinearity of the OHC contributes minimally to mechanical distortion. That is, in vivo, at moderate sound pressure levels and below, the dominant factor which contributes to nonlinearities of the OHC mechanical response resides within the nonlinear, voltage-generating, stereociliar transduction process.

Animals↗

PubMed1993

Mapping the distribution of the outer hair cell motility voltage sensor by electrical amputation.

The outer hair cell (OHC) possesses a nonlinear charge movement whose characteristics indicate that it represents the voltage sensor responsible for OHC mechanical activity. OHC mechanical activity is known to exist along a restricted extent of the cell's length. We have used a simultaneous partitioning microchamber and whole cell voltage clamp technique to electrically isolate sections of the OHC membrane and find that the nonlinear charge movement is also restricted along the cell's length. Apical and basal portions of the OHC are devoid of voltage sensors, corresponding to regions of the cell where the subsurface cisternae and/or the mechanical responses are absent. We conclude that the physical domain of the motility voltage sensor corresponds to that of the mechanical effector and speculate that sensor and effector reside within one intra membranous molecular species, perhaps an evolved nonconducting or poorly conducting voltage-dependent ion channel.

Animals↗

PubMed1993

Voltage-dependent ionic conductances of type I spiral ganglion cells from the guinea pig inner ear.

Type I spiral ganglion cells provide the afferent innervation to the inner hair cells of the mammalian organ of Corti and project centrally to the cochlear nucleus. While single-unit studies conducted over the past several decades have provided a wealth of information concerning the response characteristics of these neurons and, to some extent, their receptor targets, little is known about the neuron's intrinsic electrical properties. These properties undeniably will contribute to the firing patterns induced by acoustic stimuli. Type I spiral ganglion cell somata from the guinea pig inner ear were acutely isolated and the voltage-dependent conductances were analyzed with the whole-cell voltage clamp. Under conditions that mimic the normal intra- and extracellular ionic environments, type I spiral ganglion cells demonstrate fast inward TTX-sensitive Na currents (whose current density varied markedly among cells) and somewhat more slowly developing outward K currents. Resting potentials averaged -67.3 mV. Under current clamp, no spontaneous spike activity was noted, but short current injections produced graded action potentials with after hyperpolarizations lasting several milliseconds. The nondecaying outward K current activated at potentials near rest and was characterized by a pronounced rectification. The kinetics of the Na and K currents were rapid. Maximum peak inward Na currents occurred within 400 microseconds, between a voltage range of -10 and 0 mV, and inactivated within 4 msec. Recovery from inactivation was also rapid. At a holding potential of -80 mV, the time constant for recovery from an inactivating voltage step to -10 mV was 2.16 msec. Above -50 mV outward K currents reach half-maximal amplitude within 1.5 msec. In addition to these currents, a slow noninactivating TTX-sensitive inward current was observed that was blockable with Cd2+ or Gd3+. Problems encountered with blocking the tremendous outward K current hampered the characterization of this inward current. Similarities between the kinetics of ganglion cell currents and some of the rapid temporal characteristics of eighth nerve single-unit activity confirm the notion that intrinsic membrane properties help shape auditory neuron responses to sound.

Action Potentials↗

PubMed1992

On the frequency limit and phase of outer hair cell motility: effects of the membrane filter.

Whole-cell voltage clamp and displacement-measuring photodiode techniques were used to study electrophysiological and mechanical properties of the guinea pig outer hair cell (OHC). OHCs demonstrate a voltage-mechanical response (V-M) function that can be fit by a two state Boltzmann relation, where the cell normally rests near the hyperpolarizing saturation region (-70 to -90 mV). The voltage at half-maximal length change (Vh) is depolarized relative to the resting potential, and this ensures that for symmetrical sinusoidal voltage stimulation about the resting potential, AC and DC mechanical responses will be generated. Analysis of OHC motility using pure tone voltage bursts from 11 to 3200 Hz demonstrates both AC and DC mechanical responses. By exploiting the frequency-dependent current-voltage phase separation that is characteristic of an RC-dominated system under voltage clamp, it is demonstrated that OHC motility follows the phase of AC transmembrane voltage and not that of current. For voltage stimulation across frequencies in the acoustic range, the motility cutoff frequency corresponds to the cutoff frequency of the imposed transmembrane voltage. Frequency cutoffs approaching 1 kHz have been measured but are clamp time constant limited. These observations are congruent with the voltage dependency hypothesis of OHC motility. In addition, the DC component of the mechanical response is shown to be frequency independent, but to decrease in magnitude disproportionately compared to the AC component as the magnitude of the driving voltage decreases. This is predicted from the form of the V-M function, whose level dependent DC nonlinearity is a consequence of the resting potential being displaced from Vh. The net effect is that the mechanical DC: AC ratio approaches zero for small AC voltages. Taken together, these findings question the ability of the OHC mechanical response to influence organ of Corti micromechanics at high acoustic frequencies where a tuned amplification of basilar membrane motion is hypothesized.

Animals↗

PubMed1991

Isolated supporting cells from the organ of Corti: some whole cell electrical characteristics and estimates of gap junctional conductance.

Whole cell voltage clamp studies were performed upon isolated and small groups of supporting cells from the guinea pig organ of Corti in order to evaluate junctional and non-junctional membrane characteristics. Single Hensen cells have an average input resistance and capacitance of 1.03 G omega and 24.9 pF, respectively. I-V functions indicate an outward K+ rectification, which is blocked by external TEA, intracellular Cs, or photo-irradiation of intracellularly injected fluorescent dye. Voltage clamping of pairs or small groups of cells indicates that supporting cells 'share' K+ channels within the syncitium. The input impedance of coupled cells was studied during uncoupling with CO2 or octanol media. As expected, coupled cells showed an increase in input capacitance and a decrease in input resistance over single cell values. Input capacitance is a more sensitive indicator of cell coupling than dc input resistance. During uncoupling, input capacitance values drop to single cell levels prior to an increase of dc input resistance to single cell levels. Modeling the results indicates that Hensen cells are well-coupled under normal conditions and may have junctional resistances with values less than 0.1% of the non-junctional resistance. The sensitivity of the supporting cell syncitium's input impedance to small changes in junctional resistance markedly influences the syncitium's RC filter characteristics, and thus may control the frequency response of sound evoked electrical activity measurable in supporting cells in vivo.

Animals↗

PubMed1991

Reversible inhibition of voltage-dependent outer hair cell motility and capacitance.

Outer hair cells (OHC) from the organ of Corti are capable of fast voltage-induced length changes (Santos-Sacchi and Dilger, 1988), suggesting that an associated voltage sensor should reside in the OHC plasma membrane. Voltage-dependent mechanical responses and nonlinear charge movement of isolated OHCs from the guinea pig were analyzed using the whole-cell voltage-clamp technique. Ionic currents in the cells were blocked. Nonlinear voltage-dependent charge movement or, correspondingly, voltage-dependent capacitance was measured with step or AC analysis. OHC movements were measured either from video or using a differential photodiode technique. Maximum charge movements up to 2.5 pC were measured in OHCs from the low-frequency region of the cochlea. Both AC and step analyses indicated a peak nonlinear capacitance of 16-17 pF. The voltage dependence was fit to a Boltzmann relation with the step analysis indicating a maximum nonlinear capacitance at -23 mV step potential from a holding potential of about -120 mV, and AC analysis indicating a maximum at a holding potential near -40 mV. AC analysis probably provides a more accurate evaluation of voltage dependence. Measures of OHC motility magnitude versus voltage follow the nonlinear capacitance-voltage function obtained from AC measures. Treatment of the cells with gadolinium ions (0.5-1 mM) blocked OHC motility. This treatment also produced a shift of the nonlinear capacitance function along the voltage axis in the depolarizing direction, which can be explained by membrane surface charge screening. However, maximum capacitance was reduced as well and may correspond to the reduction or abolition of OHC motility in response to gadolinium treatment. Gadolinium effects were reversible. Nonlinear capacitance is not a function of membrane deformation due to length changes, since removal of OHC cytosol via the patch pipette abolished longitudinal movement but did not reduce nonlinear charge movement. It is interesting to note that the nonlinear capacitance will dynamically influence the time constant of the OHC during acoustically evoked receptor potential generation.

Animals↗

PubMed1989

Asymmetry in voltage-dependent movements of isolated outer hair cells from the organ of Corti.

The electrically induced movements of outer hair cells (OHC) were studied using the whole-cell voltage-clamp technique and video analysis. Cell shortening occurs during depolarization and elongation occurs during hyperpolarization from holding potentials near -70 mV. However, a marked asymmetry in response magnitude exists such that depolarization produces larger cell length changes than do comparable levels of hyperpolarization. The response is such that at normal resting potentials in vivo, displacements are about 2 nm/mV, but increase to about 15 nm/mV as the cell is depolarized. This mechanical rectification in the depolarizing direction manifests itself during symmetrical sinusoidal voltage stimulation as a "DC" reduction in cell length superimposed upon "AC" length changes. The observed OHC mechanical rectification may be involved in the reported production of "DC" basilar membrane displacements during suprathreshold acoustic stimulation (LePage, 1987). Estimates of the magnitude of OHC movements at acoustic threshold levels induced by receptor potentials in the high-frequency region of the cochlea indicate a disparity between basilar membrane and OHC movements on the order of 21 dB. Thus, it appears questionable whether OHC mechanical movements solely underlie the "active process" thought to be responsible for the high degree of neural tuning at sound pressures near 0 dB.

Animals↗

PubMed1988

Whole cell currents and mechanical responses of isolated outer hair cells.

Outer hair cells (OHCs) exhibit electrically induced cell movements which are considered to enhance the frequency selectivity and sensitivity of basilar membrane vibration. Using simultaneous whole cell voltage clamp and video analysis, we demonstrate that the mechanical response of OHCs is not altered by agents which alter membrane currents under voltage clamp. Thus the underlying mechanism of OHC movements appears to be dependent upon membrane potential, rather than transmembrane currents.

Animals↗

PubMed1987

Electrical coupling differs in the in vitro and in vivo organ of Corti.

Electrical communication between the supporting cells of the guinea pig organ of Corti was studied. For in vitro experiments, the inner ear was rapidly removed and placed in a heated perfusion chamber. Medium 199 was used. The bony cochlea and the lateral wall (spiral ligament and stria vascularis) were removed to expose the top two coils of the organ of Corti. In vivo experiments were performed upon anesthetized animals whose cochleas were exposed surgically. A tiny fenestra was made in the bony cochlea which permitted the passage of electrodes through the lateral wall and into the organ of Corti of the third turn. Coupling was assessed by impaling neighboring cells with 3 M KCl electrodes, and noting the spread of intracellularly injected current. Coupling ratios in the in vitro preparation were consistently greater than those obtained in vivo (0.58 +/- 0.17 vs. 0.104 +/- 0.064). Differences exist between the in vitro and in vivo preparations which might account for these results. In vivo the supporting cells are bathed in two different media, endolymph apically, and perilymph basally. Consequently, on their apical side the supporting cells are exposed to fluid high in K+, low in Ca2+ and at a potential of 80 mV, the endolymphatic potential. In vitro the cells are bathed on all sides in fluid similar to perilymph. Intermixing the fluids in an in vivo preparation, by tearing away the stria vascularis and Reissner's membrane, increases the magnitude of the coupling ratio (0.455 +/- 0.209). Thus the unique microenvironment of the inner ear maintains lower coupling ratios, and smaller space constants for the supporting cells.

Animals↗

PubMed1986

Dye coupling in the organ of Corti.

Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios greater than 0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.

Animals↗

PubMed1986

Immunocytochemical demonstration of astrocytes in the eighth cranial nerve of the red-eared turtle.

Coronal sections through the eighth cranial nerve and medulla of the red-eared turtle were immunocytochemically stained for the astrocytic intermediate filament protein, glial fibrillary acidic protein. Reaction product appeared in the medial one-third of the eighth cranial nerve fibers as they penetrated the medulla. The remaining two-thirds of the nerve did not show any staining for glial fibrillary acidic protein. The lateral aspect of the nerve corresponds with the extracranial portion of the nerve. When the eighth cranial nerve of the turtle has been transected extracranially it still regenerates into the cochlear nuclei. Results of the present study indicate that the eighth cranial nerve was transected in earlier studies in a region devoid of astrocytes.

Animals↗

PubMed1986

The temperature dependence of electrical coupling in the organ of Corti.

Electrical coupling in an in vitro preparation of the organ of Corti was evaluated during changes in temperature of the bathing media. The effect of cooling the organ from 35 +/- 2 degrees C to 17 +/- 3 degrees C is to reduce membrane potentials, increase input resistance and decrease coupling ratios. Decreases in coupling ratios ranging from 15 to 75% have been observed. The effects are reversible upon warming. Membrane potentials are very susceptible to depolarization caused by cooling. The reduction in coupling is not due to depolarization nor is it dependent upon extracellular Ca2+. It is conceivable, however, that intracellular stores of Ca2+ are released or that intracellular pH is altered.

Animals↗

PubMed1985

The effects of cytoplasmic acidification upon electrical coupling in the organ of Corti.

The supporting cells of the organ of Corti are joined to one another by gap junctions, and electrical coupling among them is known to be good. It is demonstrated here, using an in vitro preparation, that electrical communication between Hensen's cells can be modified by treatments which are known to cause cytoplasmic acidification. Treatment of the preparation with 100% CO2-saturated medium causes a drop in membrane potential, increase in input resistance, and decrease in steady-state coupling ratio. These measures return to pretreatment levels upon washout of the CO2 medium. Also, direct injection of H+ into a Hensen's cell uncouples that cell from the supporting cell network. An increase in coupling ratio is sometimes observed immediately before and after uncoupling due to CO2 treatment. In fact, in some cases it is possible to solely increase coupling ratios with limited CO2 treatments, although prolonged treatment with CO2 invariably produces uncoupling. This phenomenon may be due to an increase in cell resistance without a change in junctional conductance. A few possible roles for gap junctions in the inner ear are suggested, and the significance of the present results discussed.

Acid-Base Equilibrium↗

PubMed1984

A re-evaluation of cell coupling in the organ of Corti.

Intercellular electrical coupling was assessed in an in vitro organ of Corti preparation using separate electrodes to inject current and record voltage drops in Hensen's cells. The results demonstrate much better coupling among these supporting cells than previously thought. Coupling ratios between adjacent Hensen's cells are greater than 0.6.

Animals↗