Marta Martini

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)

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.

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE1 (prostaglandin E1). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF1α was less effective and neither PGE2 nor PGF2α had any effect. Dopamine and PGE, alone or in combination, did not modify low Km cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μM), which increased cyclic AMP as much as 10 μm‐dopamine, showed no synergistic effect when tested in the presence of PGE1 or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE1 Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE1 as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE1

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.

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.

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.

Potassium currents in the hair cells of vestibular epithelium: position‐dependent expression of two types of A channels

The complement of voltage‐dependent K+ currents was investigated in hair cells of the frog crista ampullaris. The currents were recorded in transversal slices of the peripheral, intermediate and central regions of the crista by applying the patch clamp technique to cells located at different positions in the slices. Voltage‐clamp recordings confirmed that cells located in each region have a distinctive complement of K+ channels. Detailed investigation of the currents in each region revealed that the complement of K+ channels in intermediate and central regions showed no variations among cells, whereas peripheral hair cells differed in the expression of two classes of A‐type currents. These currents showed different kinetics of inactivation as well as steady‐state inactivation properties. We termed these currents fast IA and slow IA based on their inactivation speed. The magnitude of both currents exhibited a significant gradient along the transversal axis of the peripheral regions. Fast IA magnitude was maximal in cells located in the external zone of the crista slice and decreased gradually to become very small in the median zone (centre) of the section, while the gradient of slow IA magnitude was reversed. A‐type currents appear to act as a transient buffer that opposes hair cell depolarization induced by positive current injections. However, fast IA is partially active at the cell resting potential, while slow IA can be recruited only following large hyperpolarizations. Thus, two types of A currents are differentially distributed in vestibular hair cells and have different roles in shaping receptor potential.