Chick Cochlear Hair Cells Research Articles

Intra- and extracellular calcium modulates stereocilia stiffness on chick cochlear hair cells.

Segments of the chick basilar papilla were isolated and maintained in culture medium. The sensory hair bundle of individual hair cells was observed with light microscopy and stimulated with a water microjet at 600 Hz. Hair bundle motion was slowed by illuminating the microscope with stroboscopic light, and water jet intensity was systematically varied in decibel (dB) steps until a visual detection level (VDL) threshold of hair bundle motion was achieved. The VDL threshold of many hair cells was measured in each isolated papilla. However, only one of eight extracellular calcium concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 1.25, 6.0, and 12.0 mM) was used with each papilla. In a second series, a calcium ionophore (ionomycin) was added to the culture medium, and VDL thresholds were again measured at seven of these extracellular calcium concentrations. With extracellular calcium alone, the stimulus level needed to achieve threshold was reduced by 2.73 dB between 0.1 and 0.01 mM. This change in threshold represented a 1.37-fold decrease in hair bundle stiffness. When ionomycin was added to the culture medium, a progressively greater stimulus intensity was needed to achieve threshold as calcium concentration increased. The 11.7-dB increase in threshold, with the addition of ionomycin, between 0.0001 and 6.0 mM extracellular calcium was equivalent to a 3.85-fold increase in bundle stiffness. These large changes in hair-bundle stiffness, as a function of the extra- or intracellular calcium environment, may play an important role in the micromechanical behavior of the hair cell during sound simulation.

Single channel recordings of calcium currents in chick cochlear hair cells.

Single Ca2+ channel currents were recorded from chick cochlear hair cells by using cell-attached patches. The elementary current was about -2 pA at 70 mV positive to the resting potential (R.P.+ 70 mV); the slope conductance over a 40 mV voltage range was 24 pS. The open time histogram was reasonably well expressed by a single exponential function, while the closed time histogram was expressed by a sum of two exponential functions. The Ca2+ channel was activated around R.P.+ 60 mV and the average ensemble current did not decay during 130 ms depolarization, suggesting that the Ca2+ channel in chick cochlear hair cells is the L-type. The Ca2+ channel has three modes of gating behaviour, expressed as current records with brief openings (mode I), no openings (mode 0), or long-lasting openings and very brief closings (mode 2).

Cisplatin blocks mechano-electric transducer current in chick cochlear hair cells

The effects of cisplatin (cis-dichlorodiammine platinum II, CDDP) on the mechano-electrical transduction (MET) current were investigated with a whole-cell patch-electrode voltage clamp technique in dissociated cochlear hair cells of chicks. CDDP blocked the MET channel in a dose-and voltage-dependent manner. At -50 mV, CDDP blocked the MET channel with a Hill coefficient of approximately 2 and a dissociation constant ( K D ) of 1.5×10 −3 M. The kinetics of CDDP blockade consist of a voltage-independent and a voltage-dependent component.

Dihydrostreptomycin modifies adaptation and blocks the mechano-electric transducer in chick cochlear hair cells

Block of the mechano-electric transduction (MET) channel by dihydrostreptomycin (DHSM) and its effects on adaptation were investigated in dissociated cochlear hair cells of the chick with a whole-cell patch-electrode voltage clamp technique. DHSM reversibly blocked the MET channel in a dose- and voltage-dependent manner. At −50 mV, DHSM blocked the MET channel with a Hill coefficient of approximately 1 and a dissociation constant ( K d) of2 × 10 −5M. Rate constants for the DHSM to bind and to unbind to and from the channel were estimated, and could be larger than5 × 10 7M −1 ·s −1 and1 × 10 3s −1, respectively. The amplitude of MET current decreased during a constant displacement of the hair bundle. This current decay, the adaptation, disappeared in the DHSM medium. The disappearance and the emergence of adaptation did not have a simple relationship with the block of MET channel by DHSM, but appeared with some delay.

The dependence of calcium-activated potassium currents on membrane potential.

Previous experiments on cholinergic synapses in chick cochlear hair cells have shown that calcium entering through acetylcholine-activated synaptic channels in turn activates calcium-dependent potassium currents, resulting in synaptic inhibition. In voltage-clamp experiments such currents would be expected to increase with depolarization (as the driving force for potassium entry is increased) and then decrease towards zero as the membrane approaches the calcium equilibrium potential (when calcium entry is suppressed). In the hair cells, however, such currents approached zero at about +20 mV, more than 170 mV negative to the calcium equilibrium potential. Another feature of the synapse is its post-junctional morphology: a uniform 20 nm cleft is formed between the postsynaptic membrane and the outermost membrane of an underlying cisterna. Here we present a model in which synaptic activation results in calcium influx into the subsynaptic cleft and thence into the bulk of the cytoplasm. The model suggests that the voltage dependence of the calcium-activated potassium current can be accounted for by only two basic assumptions: (i) entry of calcium through the activated synaptic channels by simple diffusion; and (ii) activation of the potassium channels by the cooperative action of four calcium ions. In addition, the model suggests that during activation the calcium concentration in the restricted subsynaptic space can reach levels adequate to activate the potassium channels, without requiring additional, more complicated, considerations (for example, secondary calcium release from the cisterna).

Stiffness of hair bundles in the chick cochlea

The stiffness of hair bundles from isolated chick cochlear hair cells was measured in tissue culture medium. A water jet was used to deflect fiberglass fibers, quartz fibers, and hair bundles of isolated hair cells. A voltage-displacement curve was generated for a water jet ramp stimulus applied to miniature fiberglass and quartz fibers. Fiber displacements were measured using video image subtraction techniques. A force-voltage calibration curve was then derived for the fibers by modelling them as cantilever beams subjected to point forces at the tips. A voltage-displacement curve was then generated for isolated hair cell stereociliary bundles using the same procedure as for the fibers. A corresponding force-displacement curve was derived for isolated hair cells under water jet stimulation by correlating maximum ramp voltage from the hair cell's voltage-displacement curve to a corresponding force applied to a fiber from the fiberglass fiber calibration curve. The stiffness of the hair bundle, which is the slope of the hair cell's force-displacement curve, was then calculated using Hooke's law, assuming the force was distributed along the entire length of the hair bundle. The mean stiffness value was 5.04 ± 2.68 × 10 −4 N/m for 14 hair cells, and was in close agreement with previously reported stiffness values of several investigators utilizing different animal models and procedures.

A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle.

Muscarinic agonists and ATP increase the intracellular Ca2+ concentration in chick cochlear hair cells.

1. Cholinergic muscarinic agonists applied by the pressure puff method increased intracellular Ca2+ concentration in Fura-2-loaded hair cells. The Ca2+ response outlasted the agonist application. 2. The Ca2+ response induced by acetylcholine (ACh) was ACh dose dependent with a KD of 200 microM. Desensitization was negligible, and almost identical Ca2+ responses were observed when two ACh puffs were separated by 150 s. The response was blocked by d-tubocurarine (dTC). The KD of dTC blocking was 500 microM when 100 microM-ACh induced the Ca2+ response. 3. The amplitude of the ACh-induced Ca2+ responses were potentiated to 3 times the control by incubation with calcitonin gene-related peptide (CGRP; 0.1-1 microM). CGRP did not affect the resting Ca2+ concentration. Glycine (100 microM) potentiated the ACh response to 1.4 times the control, and also increased the resting Ca2+ concentration slightly. 4. The ACh-induced Ca2+ response was suppressed by atropine. It was induced in Ca2(+)-free extracellular medium, and in Ca2(+)-free medium desensitization to a second ACh stimulation was significant. The amplitude of the second Ca2+ response was 44% of the first when two ACh puffs were separated by 117 s in Ca2+ free medium. 5. Muscarine and carbamylcholine induced similar Ca2+ responses, with KD values of 130 microM for muscarine and 340 microM for carbamylcholine. Desensitization of Ca2+ responses was negligible in both agonists. 6. ATP co-exists with ACh in some presynaptic nerve terminals (Burnstock, 1981). Puff-applied ATP (100 microM) generated a Ca2+ response with a rapid rising phase and a following slow phase. In Ca2(+)-free medium the rapid phase disappeared and only the slow phase was observed. The rapid phase is due to the influx of Ca2+ ions and the slow phase is due to a release of Ca2+ ions from an intracellular reservoir. Under voltage clamp ATP induced a fast inward current and a following slow outward current. 7. Nicotine, adenosine, glycine, GABA, glutamate and bradykinin did not induce Ca2+ responses in the hair cell. 8. ACh induced hyperpolarization of the hair cell membrane under current clamp, most probably by the activation of Ca2+ activated K+ conductance. Therefore, a cholinergic muscarinic receptor may mediate the inhibitory effects of efferent innervation observed in hair cells.

Muscarinic agonists and ATP increase intracellular Ca concentration and induce electrical responses in chick cochlear hair cells

Muscarinic agonists and ATP increase intracellular Ca concentration and induce electrical responses in chick cochlear hair cells

Muscarinic agonists and ATP increase intracellular Ca concentration and induce electrical responses in chick cochlear hair cells

Muscarinic agonists and ATP increase intracellular Ca concentration and induce electrical responses in chick cochlear hair cells