The presynaptic function of mouse cochlear inner hair cells during development of hearing

Abstract

Before mice start to hear at approximately postnatal day 10, their cochlear inner hair cells (IHCs) spontaneously generate Ca(2+) action potentials. Therefore, immature IHCs could stimulate the auditory pathway, provided that they were already competent for transmitter release. Here, we combined patch-clamp capacitance measurements and fluorimetric [Ca(2+)](i) recordings to study the presynaptic function of IHCs during cochlear maturation. Ca(2+)-dependent exocytosis and subsequent endocytic membrane retrieval were already observed near the date of birth. Ca(2+) action potentials triggered exocytosis in immature IHCs, which probably activates the auditory pathway before it becomes responsive to sound. IHCs underwent profound changes in Ca(2+)-channel expression and secretion during their postnatal development. Ca(2+)-channel expression increased toward the end of the first week, providing for large secretory responses during this period and thereafter declined to reach mature levels. The efficacy whereby Ca(2+) influx triggers exocytosis increased toward maturation, such that vesicle fusion caused by a given Ca(2+) current occurred faster in mature IHCs. The observed changes in Ca(2+)-channel expression and synaptic efficacy probably reflected the ongoing synaptogenesis in IHCs that had been described previously in morphological studies.

Fig. 1.

Ca²⁺-dependent exocytosis can be evoked by depolarizations long before the onset of hearing.A shows [Ca²⁺]_i(top panel) and C_m(bottom panel) of an IHC from a P2 mouse at low time resolution. In response to long depolarizations (200 and 500 msec), sizable increases in C_m (exocytosis) and in [Ca²⁺]_i were observed. Shorter depolarizations resulted in much smaller responses (arrow, 50 msec). The subsequent decline inC_m most likely reflects endocytic membrane retrieval. B, C_m (top panel) and membrane current (bottom panel) of an IHC from a P6 mouse in response to 100 msec long depolarizations to −5 mV in the presence or absence of extracellular Ca²⁺ (Ca²⁺-free Ringer containing 2 mm Ca²⁺-free EGTA and 3 mm MgCl₂).C_m increments were inhibited by abolition of the Ca²⁺ influx and restored on readdition of Ca²⁺. C, Representative Ca²⁺ currents (bottom panel) and C_m increments (top panel) of IHCs from different developmental stages to a 50 msec long depolarization to −5 mV. Baseline capacitance values are indicated below each trace.

Fig. 2.

Developmental changes in Ca²⁺current and exocytosis. A demonstrates the increase in basal cell capacitance during the course of maturation (P0,n = 13; P2, n = 2; P4,n = 10; P6, n = 10; P10,n = 9; and P14–P25, n = 28).B displays normalized ΔC_m-responses (top panel) to 20 or 100 msec long depolarizations to −5 mV (number of cells as in A, normalized to the average responses of P6 cells to either depolarization). The bottom panel shows the Ca²⁺ current densities for 20 and 100 msec depolarizations (peak Ca²⁺ currents normalized to the cell capacitance). The arrow indicates the onset of hearing. The diagrams depict the preferential abundance of multiple spherical bodies at the active zones of immature IHCs and the typical presence of only one plate-like ribbon at the mature afferent synapse. C displays representative current voltage relationships for three different developmental stages after subtracting the leak current (estimated by a linear fit to the hyperpolarized portion of the data).

Fig. 3.

Changes in secretion kinetics during maturation. Secretion kinetics was investigated by measuring ΔC_m to depolarizations (to −5 mV) of different duration applied in random order. A plots the mean C_m responses recorded in the perforated-patch configuration in IHCs from the different stages (n = 13 cells for P0; n = 10 for P6; n = 9 for P10; and n = 28 for IHCs from hearing mice). B displays the first 30 msec of stimulation with higher resolution. C compares the ΔC_m of whole-cell experiments on IHCs from P6 and hearing mice in which we added a mixture of 5 mm Ca²⁺-free EGTA and 5 mmCa²⁺-loaded EGTA to the pipette (n = 9 cells for P6; n = 27 for IHCs from P14–P25). The solid lines represent double exponential fits to the data to estimate size and release time constant of the readily releasable pool (see text).

Fig. 4.

Ca²⁺ dependence of exocytosis during and after maturation. The plot relates secretory responses of 5 mm EGTA-buffered IHCs from P6 and P14–P25 mice to their Ca²⁺ current integrals (restricted to small integrals; n = 9 cells for P6 andn = 27 for P14–P25 IHCs). The dashed lines represent linear fits to the first three points of each data set.

Fig. 5.

Spontaneous action potentials trigger exocytosis and increase [Ca²⁺]_i in immature IHCs.A displays an AP train recorded in the whole-cell configuration in the absence of any holding current from a representative P6 IHC. In this particular cell, spontaneous activity was observed during the first 260 sec of the whole-cell recording.B displays two spontaneous APs from the same cell at higher time resolution. C shows a low-time resolution plot of [Ca²⁺]_i and membrane potential (V_m) of an IHC in which we applied 10 pA of depolarizing current for short intervals (indicated bybars) after spontaneous activity had ceased.D shows an average of three secretory responses of two IHCs from a P6 mouse to a single action potential. We used the voltage-clamp mode to measure capacitance before and after an AP-like voltage-waveform to −10 mV. The time periods ofC_m measurements can be recognized from the band-like (sinusoidal) signals in the voltage and currenttraces.