Bruna Pelucchi

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)

Calcium‐activated potassium current clamps the dark potential of vertebrate rods

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of ≈ −35 mV. The present work investigates the physiological role of the Ca2+‐activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light‐ and dark‐adapted rods under voltage‐ or current‐clamp conditions, respectively. In light‐adapted rods of the salamander, selective blockade of Ca2+‐activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current‐clamped rods by ≈ 30 mV, from a resting potential of ≈ −35 mV. A similar depolarization was observed if external Ca2+ (1 mm) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to −100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of ≈ −20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark‐adapted current‐clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady‐state depolarizations of ≈ 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+‐activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.

Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation.

In salamander rods, Ca2+‐activated K+ current (IKCa) provides an effective “clamp” of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large‐conductance Ca2+‐ and voltage‐dependent potassium (BK) channel and intermediate‐conductance Ca2+‐dependent potassium (IK) channel, but not small‐conductance Ca2+‐dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net IKCa to 36% and 63%, respectively, whereas the current was unaffected by application of 1 μM apamin. Consistently, anti‐ SK1, ‐SK2, and ‐SK3 antibodies were unable to stain rod photoreceptors, whereas both anti‐BK and ‐SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current‐clamp experiments, it was clearly seen that the strong clamping effect of the total IKCa was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

The effect of clofilium, a K-channel blocker, on the electrogenic K secretion and the sensory discharge at the frog semicircular canal

Potassium transport by dark cells produces marked K-concentration differences between endo- and perilymphatic fluids in labyrinthine organs and generates the transepithelial potential. The ensuing electrochemical potential for K sustains the transduction current which regulates activity at the cytoneural junction. Clofilium, a compound which is known to block cardiac K channels and to decrease the endocochlear potential, was applied to the endolymphatic side of the isolated frog semicircular canal. The drug abolished the transepithelial potential and increased K outflux from the lumen to the dark cells (or the basolateral perilymph) with no apparent interference with active K secretion. When applied to the perilymphatic side in the intact labyrinth, clofilium reduced the rate of occurrence of miniature excitatory postsynaptic potentials (mEPSPs), both at rest and in response to mechanical stimulation (sinusoidal rotation at 0.1 Hz, 12.5 deg/s2 peak acceleration). This effect may be related to a reduced K-electrochemical unbalance and a decreased transduction current. The drug consistently reduced mEPSP size, although amplitude distributions remained log-normal and time intervals between successive mEPSPs remained exponentially distributed; this suggests a direct effect of clofilium on the postsynaptic membrane, in addition to any possible presynaptic effects. Spike discharge by the afferent fibre was almost completely abolished at rest and responses to mechanical stimulation were reduced by 85-90%. These effects cannot be accounted for by the mild reduction of mEPSP rates and confirm a direct action of clofilium on the afferent postsynaptic terminal.

The effects of perilymphatic tonicity on endolymph composition and synaptic activity at the frog semicircular canal

The effects of changes in perilymphatic tonicity on the semicircular canal were investigated by combining the measurements of transepithelial potential and endolymphatic ionic composition in the isolated frog posterior canal with the electrophysiological assessment of synaptic activity and sensory spike firing at the posterior canal in the isolated intact labyrinth. In the isolated posterior canal, the endolymph was replaced by an endolymph-like solution of known composition, in the presence of basolateral perilymph-like solutions of normal (230 mosmol/kg), reduced (105 mosmol/kg, low NaCl) or increased osmolality (550 mosmol/kg, Na-Gluconate added). Altered perilymphatic tonicity did not produce significant changes in endolymphatic ionic concentrations during up to 5 min. In the presence of hypotonic perilymph, decreased osmolality, K and Cl concentrations were observed at 10 min. In the presence of hypertonic perilymph, the endolymphatic osmolality began to increase at 5 min and by 10 min Na concentration had also significantly increased. On decreasing the tonicity of the external solution an immediate decline was observed in transepithelial potential, whereas hypertonicity produced the opposite effect. In the intact frog labyrinth, mEPSPs and spike potentials were recorded from single fibers of the posterior nerve in normal Ringers (240 mosmol/kg) as well as in solutions with modified tonicity. Hypotonic solutions consistently decreased and hypertonic solutions consistently increased mEPSP and spike frequencies, independent of the species whose concentration was altered. These effects ensued within 1-2 min after the start of perfusion with the test solutions. In particular, when the tonicity was changed by varying Na concentration the mean mEPSP rate was directly related to osmolality. Size histograms of synaptic potentials were well described by single log-normal distribution functions under all experimental conditions. Hypotonic solutions (105 mosmol/kg) markedly shifted the histograms to the left. Hypertonic solutions (380-550 mosmol/kg, NaCl or Na-Gluconate added) shifted the histograms to the right. Hypertonic solutions obtained by adding sucrose to normal Ringers solution (final osmolality 550 mosmol/kg) increased mEPSP and spike rates, but did not display appreciable effects on mEPSP size. All effects on spike discharge and on mEPSP rate and size were rapidly reversible. In Ca-free, 10 mM EGTA, Ringers solution, the sensory discharge was completely abolished and did not recover on making the solution hypertonic. These results indicate that perilymphatic solutions with altered tonicity produce small and slowly ensuing changes in the transepithelial parameters which may indirectly affect the sensory discharge rate, whereas relevant, early and reversible effects occur at the cytoneural junction. In particular, the modulation of mEPSP amplitude appears to be postsynaptic; the presynaptic effect on mEPSP rate of occurrence is presumably linked to local calcium levels, in agreement with previous results indicating that calcium inflow is required to sustain basal transmitter release in this preparation.

Effects of clofilium, a K channel blocker, on electrogenic K secretion and afferent discharge at the frog semicircular canal. A preliminary report.

Application of clofilium to the endolymphatic side of the isolated frog semicircular canal abolished the transepithelial potential and produced increased K and mannitol outfluxes from the lumen to the dark cells or the basolateral perilymph, with no apparent effect on active K secretion. These results suggest an increased permeability of the paracellular pathway. When applied to the perilymphatic side in the intact labyrinth, clofilium reduced the rates of quantal transmitter release (miniature EPSP frequency), an effect that might arise from a decrease in the transduction current intensity secondary to the reduced transepithelial electrochemical potential for K+. Moreover, afferent spike rates were almost completely abolished at rest as well as during mechanical stimulation. This effect together with a decreased mEPSP amplitude points to a further direct action of clofilium on the afferent postsynaptic terminal. These results suggest a multi-factorial effect of clofilium that would reduce the sensitivity of the vestibular function.