Sound-evoked spikes in the auditory nerve can phase-lock with submillisecond precision for continuous periods of time. sinewave stimulus. This similarity was explained by an unexpected getting: large-amplitude multiquantal EPSCs have a significantly larger synchronization index than smaller evoked EPSCs. Large EPSPs consequently enhance the precision of spike timing. The hair cells’ unique capacity for continuous large-amplitude and highly synchronous multiquantal launch therefore underlies its ability to result in phase-locked spikes in afferent materials. recordings of EPSPs and spikes evoked by sinusoidal stimuli that mimic pure tone sounds recapitulated several important features of recordings of afferent dietary fiber spikes. Counter-intuitively we find that large multiquantal EPSC events are better phase-locked than small evoked EPSCs. Large multiquantal EPSPs Limonin produced by the coincident launch of more than 4-5 quanta therefore enhance the precision of spike timing. By filtering out small and less exactly timed EPSPs the hair cell synapse promotes the precise phase-locking of afferent dietary fiber spikes to incoming sound waves. Results Hair cell resonant frequencies Hair cells are tightly imbedded in the epithelia of hearing organs. We obtained access to bullfrog amphibian papilla hair cells by cracking open the epithelium in the middle region (Number 1A; Keen and Hudspeth 2006 The amphibian papilla is definitely structured tonotopically from its rostral to caudal end for acoustic stimuli that range in rate of recurrence from 100 to 1250 Hz (Lewis et al. 1982 Recordings from turtle and frog hair cells reveal that they are electrically tuned Limonin (Crawford and Fettiplace 1980 Pitchford and Ashmore 1987 To determine their characteristic rate Limonin of recurrence (spike rate is therefore similar to the median spontaneous spike rate of 8.6 Hz for frog auditory nerve materials (Christensen-Dalsgaard et al. 1998 Some afferent dietary fiber recordings also displayed copious spontaneous extracellular EPSPs (eEPSPs). In 11 materials the signal-to-noise percentage was excellent permitting us to clearly detect and analyze the individual eEPSPs (normal eEPSP and spike rate of recurrence were 87.4 ± 76.4 Hz and 2.0 ± 2.3 Hz respectively; Number 1C1). Spikes were always triggered right after one large eEPSP or after 2 to 3 3 closely timed eEPSPs suggesting that they are Limonin all evoked by eEPSPs. However spikes were much less frequent than eEPSPs so the vast majority of eEPSPs failed to result in spikes. Similar findings are reported for adult turtle and young rat afferent materials (Yi et al. 2010 Schnee et al. 2013 but more mature rat materials appear to spike for nearly every EPSP event (Geisler 1997 Siegel 1992 Rutherford et al. 2012 To calculate the input resistance (Rinput) of the afferent materials we made whole-cell patch-clamp recordings having a potassium-based internal solution. We then injected negative step currents (50 to 250 pA) to the dietary fiber under current-clamp and the steady-state voltages were measured for those methods and plotted against the current amplitude. From your slope of a linear match to the data we acquired Rinput = 148 ± 64 MΩ (n=6). This relatively low input resistance of the afferent dietary fiber explains in part why small amplitude EPSPs are unable to result in spikes. Under whole-cell current-clamp we next IL1B analyzed the EPSPs and spikes. The resting membrane potential (Vrest) of the afferent materials was ?69.8 ± 0.7 mV (n=6; Number 1D) a similar value to rat auditory afferents (Yi Limonin et al. 2010 Rutherford et al. 2012 The average EPSP rate of recurrence was 80.5 ± 55.8 Hz (n=7) and the spike frequency 7.4 ± 9.8 Hz (n=7) neither of which is significantly different from the value from cell-attached recordings (p>0.05 unpaired Student’s hair cell synapses Given that synaptic delays vary for different levels of presynaptic depolarization how is spike phase invariance founded for sounds of different intensities? To explore this query we stimulated hair cells with sinusoidal voltage commands similar to those that hair cells encounter (Russell and Sellick 1983 Auditory hair cells have an resting membrane potential of about ?55 mV and their voltage responses to a pure tone sound follow a sinusoid with amplitudes of up to 20 mV peak-to-peak (Crawford and Fettiplace 1980 Corey and Hudspeth 1983 Holt and Eatock 1995 Therefore we used sinusoidal voltage commands centered at ?55 mV with peak-to-peak amplitude up to 20 mV. Number 4A shows Limonin the stimulus template: we 1st stepped the hair cell potential from ?90 mV to ?55 mV for 50 ms and then a sinusoidal.