Harunori Ohmori

Control of intracellular calcium by ATP in isolated outer hair cells of the guinea‐pig cochlea.

1. Intracellular calcium levels were monitored in isolated outer hair cells of the guinea‐pig cochlea using the calcium‐sensitive dye Fura‐2. 2. The calcium in the cells was studied during application of ATP externally applied from a pipette. ATP induced a rise of intracellular calcium which could be separated into two components: a rapid rise, peaking in 20 s, localized around the apical end of the cell, and a slower rise, peaking in 50‐150 s but spread throughout the cell. The effects were observed with 5, 25 and 100 microM‐ATP concentrations. 3. In the absence of external Ca2+, ATP was still able to trigger a rise in Ca2+, but with a longer delay. Under these conditions, the cells did not show the initial rapid Ca2+ rise. The result suggests that ATP can mobilize intracellular stores. 4. A rise in intracellular Ca2+ was also observed when 5 mM‐caffeine was applied to the bath. 5. Simultaneous measurements were made of whole‐cell currents and intracellular calcium. ATP activated an inward current at resting potentials of ‐60 mV. Internal Ca2+ levels increased during the inward current. In current‐clamped cells Ca2+ levels also increased during the associated depolarization produced by ATP. 6. Adenosine (150 microM) did not produce any measurable inward current. Acetylcholine (ACh, 100 microM‐1 mM) produced only a small rise in Ca2+. However, applied simultaneously with ATP, ACh suppressed the rise in intracellular Ca2+ produced by ATP, with the kinetics of a competitive antagonist. 7. Intracellular Ca2+ increased with step depolarizations of the cell above ‐20 mV during whole‐cell clamp. Large rises in Ca2+ were also observed on depolarizing the cell with isotonic KCl. 8. Calcium levels in supporting cells of the organ of Corti were sensitive to ATP. In these cells, rises in intracellular Ca2+ did not require the presence of extracellular Ca2+. 9. It is concluded that the organ of Corti contains receptors for ATP on a variety of the cells. ATP controls a direct entry of Ca2+ through the membrane and also may mobilize intracellular stores.

Acetylcholine increases intracellular Ca2+ concentration and hyperpolarizes the guinea-pig outer hair cell

Extracellularly applied acetylcholine (ACh) induced outward currents in isolated outer hair cells of a guinea-pig cochlea. The ACh induced current was carried by K+ ions. The current amplitude was ACh dose dependent with a KD of 12 microM. The ACh induced outward current was reversibly blocked by extracellularly applied atropine (1 microM), d-tubocurarine (d-TC, 1 microM), apamin (1 microM) and strychnine (0.1-10 microM). D-TC (10 microM) not only blocked the ACh induced outward current, but also reduced the amplitude of depolarization induced outward current. ACh induced a rise of intracellular Ca2+ concentration ([Ca2+]i). D-TC (10 microM) reduced but did not totally block the increase of [Ca2+]i. In a low Ca2+ (0.1 mM) extracellular medium, the amplitude of ACh induced current was reduced rapidly and was recovered gradually to the normal level after the extracellular Ca2+ concentration was resumed. It is probable that ACh hyperpolarizes the guinea-pig outer hair cell membrane by activation of a Ca(2+)-activated K+ conductance.

Synaptic responses to mechanical stimulation in calyceal and bouton type vestibular afferents studied in an isolated preparation of semicircular canal ampullae of chicken.

SummaryRelationships between the response patterns of semicircular canal afferents to mechanical stimulation and the morphologies of their peripheral endings were investigated in an isolated preparation of the anterior semicircular canal ampulla of chicken, using a combination of electrical recording with intracellular injections of Lucifer Yellow CH. The hair bundle mechanical stimulus was applied in a diffuse manner by a glass rod vibrating in the nearby bathing medium. Two types of spike discharge patterns and postsynaptic potentials were recorded. One type was found exclusively in the bouton type afferent and demonstrated a phasic increase of firing frequency and transient depolarizing postsynaptic potentials at the beginning of mechanical stimulation. These synaptic potentials were also observed spontaneously and their amplitudes were increased by membrane hyperpolarization. The other type was found exclusively in afferents with calyceal endings and showed a tonic increase of spiking frequency and depolarizing DC postsynaptic potentials with superimposing AC responses at the frequency of the mechanical stimulation. Amplitudes of postsynaptic potentials were increased by hyperpolarization. Hair cells generated depolarizing DC transduction potentials superimposed with AC potentials at frequency of the mechanical stimulation. The spontaneous spike discharging patterns of afferent nerve fibres were classified either as a regular type (CV < 0.10) or as an irregular type (CV > 0.25) on the basis of coefficient of variation (CV) of interspike intervals. The spontaneous firing rate of regular units was higher than that of irregular units. Several membrane characteristics are different between these two types of afferent fibers; irregular units had short membrane time constants and fast spikes associated with clear spike-afterhyperpolarization. These features fit with the fact that irregular units tend to have phasic responses to mechanical stimulation while regular units typically have tonic responses. Irregular units had bouton endings with an average axonal diameter thicker than the regular units which had calix endings.

Activation of glutamate receptors in response to membrane depolarization of hair cells isolated from chick cochlea.

1. Experiments were performed to identify the neurotransmitter released from hair cells of chick cochlea. An isolated hair cell was closely apposed to a cultured granule cell of the rat cerebellum, and both cells were whole‐cell voltage clamped by utilizing a nystatin perforated patch technique. 2. Depolarization of hair cells to potentials more positive than ‐20 mV induced currents in the granule cell in a 10 mM Ca2+ extracellular medium. Amplitudes of induced currents were dependent on the membrane potential of granule cells and showed an outward‐going rectification. The induced current in granule cells was reversibly suppressed by a local application of 2‐amino‐5‐phosphonovalerate (APV), which indicates that the current was generated through the activation of an NMDA subtype of the glutamate receptor expressed on the granule cell. 3. The current amplitude of the granule cell was dependent on the size of hair cell depolarization. The size of current induced in a granule cell held at +55 mV was progressively increased with hair cell depolarization from ‐20 to +10 mV. At more positive potentials, the current amplitude was decreased. This voltage dependence was similar to but did not exactly match that of Ca2+ currents in the hair cell. The granule cell current appeared at more positive membrane potentials than the Ca2+ current in hair cells. 4. When intracellular Ca2+ concentration was increased by UV irradiation of the hair cell loaded with a caged Ca2+ compound, nitr‐5, the closely apposed granule cell generated an outward current when voltage clamped at +55 mV. 5. These observations (paragraphs 2‐4) imply that the most likely neurotransmitter released from the hair cell at its synapse with the afferent nerve terminal is glutamate.

Voltage-gated and chemically gated ionic channels in the cultured cochlear ganglion neurone of the chick.

1. Electrophysiological properties of ionic channels of isolated or cultured cochlear ganglion (CG) neurones from chick embryo were studied under voltage‐clamp conditions using a patch electrode. 2. Tetrodotoxin‐sensitive Na+ current was activated by a step depolarization more positive than ‐40 mV, and was inactivated rapidly. 3. Outward‐going K+ current was activated by step depolarization to membrane potentials more positive than ‐62 mV. 4. Two types of Ca2+ currents were demonstrated, an inactivating and a non‐inactivating type. The inactivating type was activated by step depolarizations more positive than ‐69 mV and was inactivated rapidly. The non‐inactivating type was activated by step depolarizations more positive than ‐52 or ‐41 mV depending on the external divalent cation species. 5. The I‐V relationship and the activation kinetics of the non‐inactivating type Ca2+ channel was shifted in a positive direction along the voltage axis by 12 mV when extracellular 2.5 mM‐Sr2+ or Ba2+ were replaced by Ca2+. This shift was not observed in the inactivating type Ca2+ channel. 6. The amplitude of peak current through the inactivating type Ca2+ channel was in the order of Ca2+ greater than Sr2+ greater than Ba2+. The order of relative permeability through the non‐inactivating type estimated from the tail current amplitude was Ba2+ greater than Sr2+ greater than Ca2+. 7. After 5 days in culture, glutamate (30 microM), aspartate (100 microM), kainate (100 microM) and N‐methyl‐D‐aspartic acid (NMDA; 100 microM) elicited ionic currents. The glutamate response was depressed by 1 mM‐Mg2+ in a voltage‐dependent manner at negative membrane potentials and was almost extinguished by amino‐phosphonovalerate (APV) (0.1 mM). The major subtype of glutamate receptor could be of the NMDA type. 8. The permeability of the NMDA receptor channel to Na+ and Li+ was estimated from the reversal potential and was 1.0 and 0.7 compared with that of Cs+, respectively. 9. Divalent cations were more permeable than the monovalent cations through the NMDA receptor channel: PCa greater than or equal to PBa greater than PSr greater than PCs.

The effect of caged calcium release on the adaptation of the transduction current in chick hair cells.

1. Intracellular Ca2+ concentration ([Ca2+]i) was raised by photolysis of a caged calcium compound, nitr‐5, and its effects on the mechano‐electrical transduction (MET) current were studied by a whole‐cell patch electrode voltage clamp technique in dissociated hair cells of a chick. Nitr‐5 was loaded into the hair cell by incubation with the membrane‐permeable form of the compound (nitr‐5 AM). 2. Photolysis of nitr‐5 by ultraviolet (UV) light irradiation induced outward currents at ‐50 mV when recorded with a KCl‐based intracellular medium without Ca2+ chelating compounds. The average amplitude of the photo‐activated outward current was 115 +/‐ 82 pA (mean +/‐ S.D., n = 5). 3. The MET current generated at ‐50 mV showed a decay after step displacement of the hair bundle. This adaptation was accelerated after UV exposure of the cell. The adaptation was further accelerated by hyperpolarization of the membrane and was eliminated in 20‐100 microM Ca2+ extracellular media. 4. The displacement‐response relationship was shifted towards the positive direction after the UV irradiation. 5. The recovery of the transducer current after step displacement of the hair bundle was accelerated after UV irradiation, for both the inward‐going MET current recorded at ‐50 mV and the outward‐going MET current at +54 mV. However, the adaptation was not observed at positive membrane potentials even after the photolysis of nitr‐5. 6. The extent of MET current decay was reduced or disappeared in 20‐100 microM Ca2+ extracellular media and the offset time course was prolonged at the membrane potential of ‐50 mV. The current decay was not observed even after the photo‐release of intracellular Ca2+ in 50‐100 microM Ca2+ extracellular media. 7. These results (paragraphs 3‐6) suggest that the MET current adaptation is accelerated by the increase of [Ca2+]i, and that Ca2+ ions entering through MET channels are essential in the development of adaptation. 8. The adaptation of the MET current was reversibly reduced in a dihydrostreptomycin (DHSM, 20‐50 microM) medium. The time course of the adaptation changes lagged the changes in the MET current amplitude. 9. The adaptation developed or disappeared with a delay of 10‐20 s after the introduction of either the normal‐Ca2+ (2.5 mM) or the low‐Ca2+ (50‐100 microM) extracellular medium, respectively. These delays in the development and the subsidence of adaptation suggest a presence of a Ca2+ buffer site intracellularly between the adaptative site and the MET channel.

Ion channels for the mechano-electrical transduction and efferent synapse of the hair cell

Auditory and vestibular information is applied to the hair cell hair bundle as mechanical energy, and is transduced into electrical energy by gating ion channels. The m-e.t. channel has a unitary conductance of 50 pS and a broad selectivity to monovalent cations and to divalent cations. Ca ions are the most permeable through the channel. The angular displacement of the hair bundle is the primary gating factor. Circumstantial evidence indicates the possibility of the direct gating of channels by the membrane deformation itself. The transduction potential activates voltage gated Ca channel and leads to the release of neurotransmitters which activate afferent neurones. Cholinergic muscarinic receptors likely mediate the inhibitory efferent innervation to the hair cell.

Synaptic bodies and vesicles in the calix type synapse of chicken semicircular canal ampullae

Calix afferent fibers generate depolarizing DC (direct current) and superimposed AC (alternating current) potentials in response to a vibrating stimulation of the hair bundle in an isolated preparation of a chicken semicircular canal ampulla. The wave form of the postsynaptic potential appears similar to the transduction potential of hair cells, which suggests electrical transmission in the calix type synapse. However, synaptic bodies and vesicles were found by EM observation instead of gap junctions in the hair cell presynaptic to the calix afferent. The number of synaptic body was 11-12/hair cell. These structures support chemical transmission in the calix type synapse.

Mechanoelectrical Transduction in Vertebrate Hair Cells

Various membrane-bound proteins function to exchange ions across the membrane. Some of these proteins are called a carrier since ionic transport must be coupled with the transport of another substances or it requires metabolic energy. The other transporting protein is called a channel and serves as a rapid and a large scale ion transport across the membrane. The channel is opened and closed by the inherent gate. Ions transported are selected by its filter function. The transport is fast and many thousands of ions are carried in milliseconds, and achieves the unitary conductance of 10 to 100 pS. The gate is the most unique function of the channel, and depending on its nature the channel becomes either voltage-dependent, agonist-dependent or achieves an energy-transducing function. The mechanoreceptor cell is therefore a cell equipped with an ion channel whose gate is mechanically opened and closed. The ionic flow regulated in this manner generates membrane-potential changes and triggers other voltage-dependent channel events. Ca channels are activated by depolarizing, transducing potentials, allow Ca ions to flow into the cell for synaptic transmission, and initiate higher order of information processing.