Potassium currents in type II vestibular hair cells isolated from the guinea-pig's crista ampullaris

Type II vestibular hair cells were isolated from cristae ampullares of guinea-pig and maintained in vitro for 2–3 h. Outward membrane currents were studied under whole-cell voltage-clamp conditions. Type II hair cells had resting potentials of about –45 mV. Depolarizing voltage steps from a holding potential of –80 or –90 mV induced time- and voltage-dependent outward currents which slowly decayed to a sustained level. Tail currents reversed at about –70 mV, indicating that the outward currents were mainly carried by potassium ions. The currents had an activation threshold around –50 mV. The transient component was completely removed by a depolarizing pre-pulse positive to –10 mV. While bath application of 4-aminopyridine (5 mM) reduced both components, extracellular tetraethylammonium (10 mM) or zero calcium preferentially diminished the sustained current. We conclude that at least two potassium conductances are present, a delayed rectifier with a relatively fast inactivation and a calcium-dependent potassium current. Depolarizing current injections induced an electrical resonance in the voltage responses, with a frequency of 25–100 Hz, larger currents causing higher frequencies.     

Gradients of expression of calcium and potassium currents in frog crista ampullaris

In the present work we studied the intraregional expression of voltage-dependent Ca2+ and K+ currents in hair cells of frog crista ampullaris. The currents were recorded in situ from sensory cells of the peripheral region, the most populated region of the crista, by using the whole-cell variant of the patch-clamp technique. Voltage-clamp recordings revealed that the calcium current (I(Ca)) and the outward potassium currents of I(A), I(K) I(KCa) types and the inward rectifier potassium current of I(K1) type exhibited a significant gradient of density (pA/pF) along the region. I(A) density was maximal in cells located at the beginning of the peripheral region and decreased gradually becoming very small at the opposite end. All the other currents showed an opposite gradient of expression. Current-clamp experiments showed that the voltage behaviour of hair cells changed in relation to cell position. Cells located at the beginning of the peripheral region showed large depolarizations from the resting potential (close to -45 mV) which are consistent with the presence of small I(K) and I(KCa), and an I(A) largely inactivated at rest. These cells also exhibited slowly developing and large hyperpolarizations that approached passive ones, due to the lack of I(K1). In contrast, cells located at the opposite side of the region showed smaller depolarizations and hyperpolarizations from the resting potential (close to -65 mV), due to the presence of large I(K) and I(KCa), and I(K1), respectively. The possible role of the intraregional variation of Ca2+ and K+ currents in both hair cell function and afferent discharge properties is discussed.     

Isolation and possible role of fast and slow potassium current components in hair cells dissociated from frog crista ampullaris

Potassium-current inactivation and recovery kinetics are pivotal in sustaining dynamic processing of time-varying sensory signals in hair cells. We report a detailed analysis of K⁺-currents in isolated hair cells from the frog crista ampullaris. The single components were dissected using a novel procedure based on their differential kinetic properties: The fast IA component exhibited two processes of inactivation removal; the persistent I _KD component (I _KV + I _KCa), unexpectedly displayed partial inactivation, removed by negative potentials with particularly slow, delayed kinetics. The physiological relevance of these observations was investigated by imposing sinusoidal membrane potential changes to mimic receptor response to hair bundle deflection. The excitatory phase elicited extra-currents (hysteresis) only if the off phase went sufficiently negative to remove IA inactivation. Native, resting hair cells are depolarised by receptor current; thus, voltage continuously modulates I_KD, whereas IA only transiently ensues when the receptor current vanishes (zero-current potential ≈−70 mV) and polarisation removes IA inactivation.     

Synapsin-like immunoreactivity is present in hair cells and efferent terminals of the toadfish crista ampullaris

The synapsins are presynaptic membrane-associated proteins involved in neurotransmitter release. They are differentially expressed in tissues and cells of the central and peripheral nervous system. In vestibular end organs of mammals, synapsin I-like immunoreactivity has been reported in efferent and afferent terminals and in afferent nerve calyces surrounding type I hair cells. In addition, synapsin I has recently been described in several non-neural cell lines. The present study was conducted to locate synapsin-like immunoreactivity in the neuronal and non-neuronal cells of the fish crista ampullaris, to examine the possibility that the non-neuronal sensory receptor cells express synapsins in vivo. Synapsin-like immunostaining was visualized by immunofluorescence detection in wholemounts of the toadfish crista ampullaris using multiphoton laser scanning microscopy and by electron microscopic visualization of post-embedding immunogold labeling. The results demonstrate that synapsin-like immunoreactivity is present in vestibular hair cells and efferent boutons of the toadfish crista ampullaris. Afferent endings are not labeled. Staining in hair cells is not associated with the synaptic ribbons, suggesting that there is an additional, non-synaptic role for the synapsins in some non-neuronal cells of vertebrates. Moreover, while the cristae of amniote and anamniote species share many functional attributes, differences in their synaptic vesicle-associated protein profiles appear to reflect their disparate hair cell populations.     

Regional analysis of whole cell currents from hair cells of the turtle posterior crista

The turtle posterior crista is made up of two hemicristae, each consisting of a central zone containing type I and type II hair cells and a surrounding peripheral zone containing only type II hair cells and extending from the planum semilunatum to the nonsensory torus. Afferents from various regions of a hemicrista differ in their discharge properties. To see if afferent diversity is related to the basolateral currents of the hair cells innervated, we selectively harvested type I and II hair cells from the central zone and type II hair cells from two parts of the peripheral zone, one near the planum and the other near the torus. Voltage-dependent currents were studied with the whole cell, ruptured-patch method and characterized in voltage-clamp mode. We found regional differences in both outwardly and inwardly rectifying voltage-sensitive currents. As in birds and mammals, type I hair cells have a distinctive outwardly rectifying current (I(K,L)), which begins activating at more hyperpolarized voltages than do the outward currents of type II hair cells. Activation of I(K,L) is slow and sigmoidal. Maximal outward conductances are large. Outward currents in type II cells vary in their activation kinetics. Cells with fast kinetics are associated with small conductances and with partial inactivation during 200-ms depolarizing voltage steps. Almost all type II cells in the peripheral zone and many in the central zone have fast kinetics. Some type II cells in the central zone have large outward currents with slow kinetics and little inactivation. Although these currents resemble I(K,L), they can be distinguished from the latter both electrophysiologically and pharmacologically. There are two varieties of inwardly rectifying currents in type II hair cells: activation of I(K1) is rapid and monoexponential, whereas that of I(h) is slow and sigmoidal. Many type II cells either have both inward currents or only have I(K1); very few cells only have I(h). Inward currents are less conspicuous in type I cells. Type II cells near the torus have smaller outwardly rectifying currents and larger inwardly rectifying currents than those near the planum, but the differences are too small to account for variations in discharge properties of bouton afferents innervating the two regions of the peripheral zone. The large outward conductances seen in central cells, by lowering impedances, may contribute to the low rotational gains of some central-zone afferents.     

Functional analysis of whole cell currents from hair cells of the turtle posterior crista

Controlled currents were used to study possible functions of voltage-sensitive, outwardly rectifying conductances. Results were interpreted with linearized Hodgkin-Huxley theory. Because of their more hyperpolarized resting potentials and lower impedances, type I hair cells require larger currents to be depolarized to a given voltage than do type II hair cells. "Fast" type II cells, so-called because of the fast activation of their outward currents, show slightly underdamped responses to current steps with resonant (best) frequencies of 40-85 Hz, well above the bandwidth of natural head movements. Reflecting their slower activation kinetics, type I and "slow" type II cells have best frequencies of 15-30 Hz and are poorly tuned, being critically damped or overdamped. Linearized theory identified the factors responsible for tuning quality. Our fast type II hair cells show only modestly underdamped responses because their steady-state I-V curves are not particularly steep. The even poorer tuning of our type I and slow type II cells can be attributed to their slow activation kinetics and large conductances. To study how ionic currents shape response dynamics, we superimposed sinusoidal currents of 0.1-100 Hz on a small depolarizing steady current intended to simulate resting conditions in vivo. The steady current resulted in a slow inactivation, most pronounced in fast type II cells and least pronounced in type I cells. Because of inactivation, fast type II cells have nearly passive response dynamics with low-frequency gains of 500-1,000 Momega. In contrast, type I and slow type II cells show active components in the vestibular bandwidth and low-frequency gains of 20-100 and 100-500 Momega, respectively. As there are no differences in the responses to sinusoidal currents for fast type II cells from the torus and planum, voltage-sensitive currents are unlikely to be responsible for the large differences in gains and response dynamics of afferents innervating these two regions of the peripheral zone. The low impedances and active components of type I cells may be related to the low gains and modestly phasic response dynamics of calyx-bearing afferents.     

Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

Afferent innervation patterns of the pigeon horizontal crista ampullaris

The vestibular semicircular canals are responsible for detection of rotational head motion although the precise mechanisms underlying the transduction and encoding of movement information are still under study. In the present investigation, we utilized neural tracers and immunohistochemistry to quantitatively examine the topology and afferent innervation patterns of the horizontal semicircular canal crista (HCC) in pigeons (Columba livia). Two hundred and eighty-six afferents from five horizontal canal organs were identified of which 92 units were sufficiently labeled and isolated to perform anatomical reconstructions. In addition, a three-dimensional contour map of the crista was constructed. Bouton afferents were located only in the peripheral regions of the receptor epithelium. Bouton afferents had the most complex innervation patterns with significantly longer and more numerous branches as well as a higher branch order than any other fiber type. Bouton fibers also contained significantly more bouton terminals than did dimorph afferents. Calyx afferents were located only in the apex and central planar regions. Calyx fibers had the largest axonal diameters yet the smallest fiber lengths and innervation areas, the fewest number of branches, the lowest branch order, and the fewest total number of terminals of all fiber types. Dimorph afferents were located throughout the central crista with afferent terminations that were larger and more complex than calyx fibers but less so than bouton fibers. Overall, the pigeon HCC morphology and innervation shares many common features with those of other animal classes.     

Thrombin stimulates Ca-channel currents in isolated frog ventricular cells

Effects of the proteolytic enzyme thrombin in the modulation of cardiac Ca-channel currents were examined in single ventricular cells from frog myocardium, using the whole-cell voltage clamp technique (Hamill et al. 1981). Application of 3.8·10^–9 M thrombin to the bath increased the peak of the Ca-channel current by 84±35% (8 cells). Hirudin (31·10^–9 M), a specific thrombin inhibitor, blocked the thrombin-induced increase of this current. The increase in the current can be made responsible for the measured positive inotropic effects of thrombin as well as for the shift of the plateau voltage of the action potentials towards more positive values.     

Muscarinic ACh receptor activation causes transmitter release from isolated frog vestibular hair cells

In the frog, vestibular efferent fibers innervate only type-II vestibular hair cells. Through this direct contact with hair cells, efferent neurons are capable of modifying transmitter release from hair cells onto primary vestibular afferents. The major efferent transmitter, acetylcholine (ACh), is known to produce distinct pharmacological actions involving several ACh receptors. Previous studies have implicated the presence of muscarinic ACh receptors on vestibular hair cells, although, surprisingly, a muscarinic-mediated electrical response has not been demonstrated in solitary vestibular hair cells. This study demonstrates that muscarinic receptors can evoke transmitter release from vestibular hair cells. Detection of this release was obtained through patch-clamp recordings from catfish cone horizontal cells, serving as glutamate detectors after pairing them with isolated frog semicircular canal hair cells in a two-cell preparation. Although horizontal cells alone failed to respond to carbachol, application of 20 microM carbachol to the two-cell preparation resulted in a horizontal cell response that could be mimicked by exogenous application of glutamate. All of the horizontal cells in the two-cell preparation responded to 20 microM CCh. Furthermore, this presumed transmitter release persisted in the presence of d-tubocurarine at concentrations that block all known hair cell nicotinic ACh receptors. The effect on the detector cell, imparted by the carbachol application to the hair cell-horizontal cell preparation, was blocked both by 2-amino-5-phosphonopentanoic acid, a selective N-methyl-D-aspartate antagonist, and the muscarinic antagonist, atropine. Thus vestibular hair cells from the frog semicircular canal can be stimulated to release transmitter by activating their muscarinic receptors.