Low extracellular TEA concentrations affect hair cell IKCa and afferent mEPSP and spike discharge in the frog semicircular canals

Compound K+ currents are recorded from hair cells isolated from frog semicircular canals by using the patch-clamp technique in the whole-cell configuration. A mix of a fast transient IA and of a delayed IKD is evoked from negative holding levels (-100 mV) that remove IA inactivation. Command steps (-30/+40 mV range) from a -40 mV holding potential only evoke IKD and the calcium current, ICa. The fast IA is separated from the slow IKD using difference-current protocols. IKD actually is a compound current due to the summation of the slow IKV and the IKCa fraction. The IKCa and IKV components of IKD are further dissected by blocking the calcium-dependent IKCa (which contributes 70% of the whole IKD) with 200 μM extracellular Cd2+, and subtracting the residual IKV from the IKD. mEPSPs, recorded in the isolated frog labyrinth from single fibres of the posterior nerve, display a high resting frequency (>100/s), which increases during canal rotation in the excitatory direction, causing single events to overlap and preventing direct evaluation of mEPSP number, waveform and size. We have developed in the past a signal processing procedure which shortens the event duration, by Wiener filtering the original signal, and makes mEPSP analysis feasible. Recently, this procedure is refined by combining the Wiener filtering routine for event detection with least-squares-errors optimization of fit and identification of possibly neglected events. The analysis yields the best fitting set of parameters (time of occurrence, t0, size factor, h, and waveform parameters,  and β) for each event; events with particularly small amplitude or aberrant waveform can be excluded as artefacts. Spikes in the tracings are evaluated and subtracted. Each spike is substituted with a suitable number of mEPSPs (generally 5), whose summation is expected to cross the threshold for generating the underlying spikes. The procedure allows to evaluate mEPSP frequencies up to 800/s. Transmitter (glutamate) release at the cytoneural junction is asynchronous and uniquantal both at rest and during rotation. We tested the effects of low extracellular TEACl (2-5 mM) on K+ current components at single dissociated hair cells and on the sensory discharge of the posterior canal in the intact labyrinth. Results indicate that IA and IKV are insensitive to TEA (n=6), while IKCa amplitude is significantly reduced, by 40 % in 2 mM (n=5) and by 60 % in 5 mM TEA (n=5). Activation-deactivation current kinetics is unaffected. IKCa plays a major role in cell repolarisation in the voltage range spanned by natural hair cell activity, given that IKV is of limited amplitude and IA only contributes when its inactivation is removed by cell hyperpolarisation due to intense inhibitory canal stimulation or under the activation of the inhibitory efferent system. Consistent with such major putative role for IKCa ‘in vivo’, a large increase in mEPSP and spike frequency is observed when the labyrinth is exposed to 5 mM TEA. This presynaptic effect is accompanied by repetitive spike activity in the afferent nerve fibre. In 2 mM TEA, repetitive spike discharge does not occur and spontaneous mEPSPs display bursts. This suggests that the hair cell membrane potential becomes unstable when the repolarising power of IKCa is impaired, and oscillations ensue in the rate of quantal transmitter release. This effect, however, does not modify the size and waveform of the elementary mEPSP and does not prevent hair cell response to mechanical stimulation. Our results indicate a full correlation between the transduction current/IKCa imbalance, and the sensory afferent information.