Maria Lisa Rossi

Calcium Currents in Hair Cells Isolated from Semicircular Canals of the Frog

L-type and R-type Ca(2+) currents were detected in frog semicircular canal hair cells. The former was noninactivating and nifedipine-sensitive (5 microM); the latter, partially inactivated, was resistant to omega-conotoxin GVIA (5 microM), omega-conotoxin MVIIC (5 microM), and omega-agatoxin IVA (0.4 microM), but was sensitive to mibefradil (10 microM). Both currents were sensitive to Ni(2+) and Cd(2+) (>10 microM). In some cells the L-type current amplitude increased almost twofold upon repetitive stimulation, whereas the R-type current remained unaffected. Eventually, run-down occurred for both currents, but was prevented by the protease inhibitor calpastatin. The R-type current peak component ran down first, without changing its plateau, suggesting that two channel types generate the R-type current. This peak component appeared at -40 mV, reached a maximal value at -30 mV, and became undetectable for voltages > or =0 mV, suggestive of a novel transient current: its inactivation was indeed reversibly removed when Ba(2+) was the charge carrier. The L-type current and the R-type current plateau were appreciable at -60 mV and peaked at -20 mV: the former current did not reverse for voltages up to +60 mV, the latter reversed between +30 and +60 mV due to an outward Cs(+) current flowing through the same Ca(2+) channel. The physiological role of these currents on hair cell function is discussed.

Quantal nature of synaptic transmission at the cytoneural junction in the frog labyrinth.

1. The mechanism of transmitter release at the cytoneural junction of the frog posterior canal was investigated by recording intracellularly subthreshold postsynaptic potentials (EPSPs), and performing a statistical analysis of time intervals and peak amplitudes. In single units EPSPs display highly variable size, so it is not clear whether they are generated by the release of single quanta of transmitter and whether large ones represent giant events, multiquantal events, or the random summation of independent unitary events. 2. In units with low resting EPSP rates, peak amplitudes and time intervals between EPSPs were measured directly. Peak amplitude histograms were continuous, unimodal and well fitted by log normal distributions. Time‐interval histograms were well described by single exponentials. 3. At high EPSP rates (either at rest or during experimental treatments), where single events overlapped extensively, peak amplitude histograms were skewed markedly towards high values. Under these conditions, the EPSP waveform was estimated by autoregressive fit to the autocorrelation of the recorded signal. The fit was used to build a Wiener filter, for sharpening the original signal, before computing time‐interval and peak amplitude histograms. This yielded consistent log normal peak amplitude distributions with no ‘excess’ skewness, similar to those obtained with low resting rates. 4. After sharpening by the Wiener filter, shoulders or small second peaks in amplitude distributions were observed only at the highest EPSP rates (> 300 s‐1). The number of ‘multiquantal’ events was reduced by Wiener filtering, and was in general consistent with the expectation that more than one independent event occurred within the duration of the single event. This suggests that the events are uniquantal, random and independent, i.e. miniature EPSPs (mEPSPs). 5. In general, peak amplitude distributions obtained with modified external Ca2+ concentration ([Ca2+]o) and/or during mechanical stimulation or under efferent activation were not significantly altered with respect to those obtained in the same units at rest. Time‐interval histograms were generally mono‐exponential at rest as well as during mechanical or efferent stimulation, and irrespective of [Ca2+]o. Resting mEPSP rate was slightly increased by elevated [Ca2+]o and reduced by low [Ca2+]o. The increase in mEPSP rate produced by mechanical excitation was depressed by both high and low [Ca2+]o, whereas both conditions enhanced mechanical inhibition. Efferent inhibition was little affected. High [Ca2+]o hastened adaptation during efferent facilitation. Low [Ca2+]o reduced peak response during facilitation, but suppressed its warning. 6. In the presence of ATP a consistent though transient increase in resting mEPSP rate was observed in about 50% of units.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptive distortions in the generator potential of semicircular canal sensory afferents

The generator potential in sensory afferents of frog crista ampullaris was extracellularly recorded from the cut end of the posterior ampullary nerve by means of suction electrodes. A servocontrolled turntable allowed suitable rotatary stimulations. The analysis of the recorded generator potential revealed a different time course from that predicted on the basis of the pendulum model. Adaptation and undershoots in the responses to velocity ramps, steps and sinusoids, were mainly responsible for the deviations, which became very evident only when fairly high acceleration rates were applied. Both adaptation and undershoots were produced presumably by the activation of an electrogenic pump, probably located in nerv terminals contacting the hair cells. In fact, the time course of the generator potential became much more consistent with the predictions from the pendulum model under treatments capable of hindering the ion pump activity.

The slow Ca(2+)-activated K+ current, IAHP, in the rat sympathetic neurone.

1. Adult and intact sympathetic neurones of the rat superior cervical ganglion maintained in vitro at 37 degrees C were analysed using the two‐electrode voltage‐clamp technique in order to investigate the slow component of the Ca(2+)‐dependent K+ current, IAHP. 2. The relationship between the after‐hyperpolarization (AHP) conductance, gAHP, and estimated Ca2+ influx resulting from short‐duration calcium currents evoked at various voltages proved to be linear over a wide range of injected Ca2+ charge. An inflow of about 1.7 x 10(7) Ca2+ ions was required before significant activation of gAHP occurred. After priming, the gAHP sensitivity was about 0.3 nS pC‐1 of Ca2+ inward charge. 3. IAHP was repeatedly measured at different membrane potentials; its amplitude decreased linearly with membrane hyperpolarization and was mostly abolished close to the K+ reversal potential, EK (‐93 mV). The monoexponential decay rate of IAHP was a linear function of total Ca2+ entry and was not significantly altered by membrane potential in the ‐40 to ‐80 mV range. 4. Voltage‐clamp tracings of IAHP could be modelled as a difference between two exponentials with tau on approximately 5 ms and tau off = 50‐250 ms. 5. Sympathetic neurones discharged only once at the onset of a long‐lasting depolarizing step. If IAHP was selectively blocked by apamin or D‐tubocurarine treatments, accommodation was abolished and an unusual repetitive firing appeared. 6. Summation of IAHP was demonstrated under voltage‐clamp conditions when the depolarizing steps were repeated sufficiently close to one another. Under current‐clamp conditions the threshold depolarizing charge for action potential discharge significantly increased with progressive pulse numbers in the train, suggesting that an opposing conductance was accumulating with repetitive firing. This frequency‐dependent spike firing ability was eliminated by pharmacological inhibition of the slow IAHP. 7. The IAHP was significantly activated by a single action potential; it was turned on cumulatively by Ca2+ load during successive action potential discharge and acted to further limit cell excitability.

Efferent control of posterior canal afferent receptor discharge in the frog labyrinth.

EPSP and spike discharges were intracellularly recorded from 90 afferent fibres of the posterior nerve in the isolated frog labyrinth and the effects of electrical activation of the efferent system were tested. Posterior canal efferent synapses were activated, via an axon reflex, by electrical shocks to the anterior-horizontal nerves. The afferent resting discharge of all fibres tested was affected by efferent stimulation: 39 units were inhibited (43%) and 51 (57%) were facilitated. The efferent system was activated with several stimulation frequencies in the 10-200 Hz range applied for different times (250 ms-10 s). By changing the stimulus parameters, inhibition did not reverse to facilitation or vice versa. Facilitation appeared within the train above a threshold frequency of about 10 Hz. The peak response was readily reached within the first second and then, with long lasting stimulation, a marked adaptation ensued. The increase in firing rate was independent of previous resting activity. The relationship between frequency and facilitatory response is described by a logarithmic function over the 30-200 Hz range tested. At the end of short trains a consistent post-stimulation after-discharge appeared, whose intensity is positively related to stimulation frequency and time. Inhibition was achieved at stimulation rates above 10 Hz, and a post-stimulation rebound discharge was evident, which was linearly dependent on previous stimulation rate. The latency values of both inhibitory and facilitatory effects were measured by taking into account either the EPSP release rate or the spike discharge modifications at all the frequencies tested. Latency proved to decrease exponentially with increasing stimulation time from a minimal value of 3 ms to a maximum of 200 ms, with minor differences between inhibition or facilitation. These long latency values, the presence of a threshold frequency and the stimulus- and frequency dependence indicate that the efferent synapses must be activated repetitively to produce detectable effects on the afferent discharge; this is in line with the discharge pattern of the efferent system fibres physiologically measured in some systems. The present results show that the dual central control of the crista ampullaris of frog posterior canal is potentially capable of setting the receptor population at a variable level of sensitivity to mechanical stimuli, with profound modifications in the canal transfer function and the spike encoding mechanism.

Regional distribution of calcium currents in frog semicircular canal hair cells

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.

IP3 receptor in the hair cells of frog semicircular canal and its possible functional role.

The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2‐aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1 mm), without affecting mEPSP size or time course. At high concentration (1 mm), 2APB is reported to block the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of group I metabotropic glutamate receptors (RS)‐3,5‐dihydroxyphenylglycine (DHPG, 0.6 mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1 mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3‐sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge.

Effectiveness of some anions in sustaining the efferent inhibition in the frog labyrinth

Efferent inhibition in the frog labyrinth is sustained by the release of acetylcholine (ACh) which opens a Cl(-)-channel in the hair cell membrane. To investigate more closely the nature of the permeability change underlying the ACh reaction, the external Cl(-) was replaced by anions of increasing hydrated size, and to test the possible role of a Cl(-)-pump in the sensory cells, drugs were applied which are known to block active cl(-) pumping in other systems. Experiments indicate that the ACh-operated inhibitory channel of the hair cell is larger than at other inhibitory synapses (or approximately 0.7 nm), while pharmacological treatments (DNP, NaN3, acetazolamide, ammonium acetate, DIDS) fail to demonstrate any active distribution of Cl(-) across the hair cell membrane.

Ca2+-dependent kinetics of hair cell Ca2+ currents resolved with the use of cesium BAPTA.

Hair cells in the frog semicircular canal, studied by the whole-cell patch-clamp technique, display three distinct Ca2+ currents: two non-inactivating components (L type and R type, the latter termed R2 in the following) and a second R type current (termed R1), which runs down first and inactivates in a Ca2+-dependent fashion. Since intracellular EGTA, up to 5 mM, did not display major effects on such inactivation, we used increasing amounts of BAPTA in the patch pipette, to control [Ca2+]i more efficiently and investigate whether modifications in [Ca2+]i at the cytoplasmic side of the channel affect the inactivation of the R1 component and in general the gating of all channel types. The results here reported show that (1) K+ currents heavily contaminate recordings obtained using high concentrations of BAPTA in its commercially available K+ salt form; (2) BAPTA Cs+ salt can be satisfactorily employed to obtain reliable recordings; (3) the kinetics of channel gating and R1-channel inactivation are indeed markedly affected by effectively buffering [Ca2+]i.

Ionic currents in hair cells dissociated from frog semicircular canals after preconditioning under microgravity conditions

The effects of microgravity on the biophysical properties of frog labyrinthine hair cells have been examined by analyzing calcium and potassium currents in isolated cells by the patch-clamp technique. The entire, anesthetized frog was exposed to vector-free gravity in a random positioning machine (RPM) and the functional modification induced on single hair cells, dissected from the crista ampullaris, were subsequently studied in vitro. The major targets of microgravity exposure were the calcium/potassium current system and the kinetic mechanism of the fast transient potassium current, I(A). The amplitude of I(Ca) was significantly reduced in microgravity-conditioned cells. The delayed current, I(KD) (a complex of I(KV) and I(KCa)), was drastically reduced, mostly in its I(KCa) component. Microgravity also affected I(KD) kinetics by shifting the steady-state inactivation curve toward negative potentials and increasing the sensitivity of inactivation removal to voltage. As concerns the I(A), the I-V and steady-state inactivation curves were indistinguishable under normogravity or microgravity conditions; conversely, I(A) decay systematically displayed a two-exponential time course and longer time constants in microgravity, thus potentially providing a larger K(+) charge; furthermore, I(A) inactivation removal at -70 mV was slowed down. Stimulation in the RPM machine under normogravity conditions resulted in minor effects on I(KD) and, occasionally, incomplete I(A) inactivation at -40 mV. Reduced calcium influx and increased K(+) repolarizing charge, to variable extents depending on the history of membrane potential, constitute a likely cause for the failure in the afferent mEPSP discharge at the cytoneural junction observed in the intact labyrinth after microgravity conditioning.