Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss

Abstract

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF(Pax2) KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF(Pax2) KO IHCs showed a normal maturation. Likewise, in BDNF(Pax2) KO membrane capacitance (ΔC(m)) as a function of inward calcium current (I(Ca)) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC(m), but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF(Pax2) KO IHCs. Maximal ΔC(m) correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF(Pax2) KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

Figure 1.

Inactivation of BDNF in the auditory system. A–D, X-Gal staining of mice carrying the Pax2-Cre transgene on a ROSA26R background (Soriano, 1999), revealed Cre activity in the mature cochlea (A, organ of Corti, OC, and spiral ganglion neurons, SG), the dorsal (but not ventral) cochlear nucleus (B, DCN, VCN) and inferior colliculus (D, IC). No staining was observed in the olivary complex (C, MSO/LSO) and in all layers of the auditory cortex (D, inset, AC). Scale bar, 200 μm; n = 4. E, F, Immunohistochemistry for BDNF (red) on cochlear sections from control (E), BDNF^Pax2 KO (F) mice at the level of SG (left) and IHCs (right). Cell nuclei were counterstained with DAPI, n = 3 mice, done in triplicate. Scale bar, 10 μm. G, Northern blot of mRNA from cochlea, IC and AC tissue of control and BDNF^Pax2 KO mice, hybridized with a probe for BDNF exon IX recognizing BDNF mRNA isoforms (1.8 kb and 4 kb; Timmusk et al., 1993). Cyclophilin (CP) was used as reference gene, n = 4 mice, done in duplicate. H, Western blot detection of BDNF in IC and AC from control and BDNF^Pax2 mice. GAPDH was used as loading control, n = 4 mice, done in duplicate.

Figure 2.

Mild impairment of hearing threshold but similar OHC function in BDNF^Pax2 KO mice. A, Average ABR thresholds (horizontal dashes) and single ear thresholds (circles and squares) for control (black dash and open circles) and BDNF^Pax2 KO mice (red dash and squares). Average ABR thresholds ± SD for click stimuli were 11.2 dB higher in ears from BDNF^Pax2 KO mice (32 dB SPL ± 9.27 SD, n = 26/52 mice/ears) than in ears from control mice (20.8 dB SPL ± 6.04 SD, n = 27/54 mice/ears, t test: p < 0.001). B, Average frequency-specific ABR thresholds ± SD confirmed a significant increase in thresholds in BDNF^Pax2 KO mice over most frequencies measured (controls: n = 27/27 mice/ear; BDNF^Pax2 KO: n = 26/26 mice/ear; two-way ANOVA: p < 0.001). C, D, DPOAE growth function ± 95% confidence interval at f2 = 11.3 kHz (C) and 2f1–f2 DPOAE thresholds ± SD (dB SPL f1) (D) in control (n = 7/12 mice/ears) and BDNF^Pax2 KO mice (n = 7/12 mice/ears), revealed mostly similar OHC thresholds and signal amplitudes in both mouse lines.

Figure 3.

Reduced exocytosis in the basal IHCs of BDNF^Pax2 KO mice. A, B, I_Ca and ΔC_m responses from adult basal turn control and BDNF^Pax2 KO IHCs. Recordings were obtained in response to 100 ms voltage steps, in 10 mV increments, from the holding potential of −81 mV. For clarity, only maximal responses are shown in A. C, The relation between Ca²⁺ entry and exocytosis in IHCs, estimated using a synaptic transfer function, was obtained by plotting ΔC_m against the peak I_Ca for voltage steps from −71 mV to that where the maximal I_Ca occurred from the I-V curves shown in B. Data were approximated using a power function: ΔC_m ∝ I_Ca^N, where N is the power. N was 1.3 ± 0.1 (n = 5) in control and 1.1 ± 0.1 (n = 4) in BDNF^Pax2 KO mice. D, Ca²⁺ currents and changes in cell membrane capacitance from apical (ap) and basal (ba) IHCs.

Figure 4.

Reduced synaptic ribbons and fibers in BDNF^Pax2 KO mice. A, Immunohistochemistry for CtBP2/RIBEYE (green) in controls and BDNF^Pax2 KO mice, shown for the midbasal turn. Cell nuclei were counterstained with DAPI; scale bar, 10 μm. B, Ribbon counts ± SD in different cochlear turns (t test: p < 0.001). Numbers of IHCs counted are given in the bars; n = 3 mice. C, Whole mount preparation of the medial turn in controls and BDNF^Pax2 KO mice labeled with antibodies against CtBP2/RIBEYE (green) and NF200 (red; top). Cell nuclei were counterstained with DAPI; scale bar, 5 μm. The lower panel displays isoprojections for the different labelings of the upper panel. D, Reduction of ABR wave I amplitude at 20 dB above hearing threshold (left) and growth function (right) in BDNF^Pax2 KO mice compared with control, mean ± SEM. Controls: n = 17/34 mice/ears; BDNF^Pax2 KO: n = 17/34 mice/ears; two-way ANOVA: p < 0.001.

Figure 5.

BDNF^Pax2 KO mice are less vulnerable to noise trauma. A, B, Mean ABR thresholds ± SD for click stimuli (A) and mean frequency-specific ABR thresholds ± SD (B), seven days after noise exposure (AT, 116 dB SPL, 10 kHz, for 40 min) in control and BDNF^Pax2 KO mice (nonexposed control: n = 6/12 mice/ears; control +AT: n = 8/16; nonexposed BDNF^Pax2 KO: n = 6/12, BDNF^Pax2 KO +AT: n = 8/16; A, one-way ANOVA: p < 0.001; B, two-way ANOVA: p < 0.001). C, Ribbon counts from three different frequency cochlear regions in sham- or noise-exposed controls and BDNF^Pax2 KO mice (one-way ANOVA: p < 0.001); n = 3 mice. Numbers of IHCs counted are given in the bars. D, E, Immunohistochemistry for CtBP2/RIBEYE (green) in controls (D) and BDNF^Pax2 KO mice (E) before and 14 d after noise exposure (+AT), shown for the midbasal turn. Cell nuclei were counterstained with DAPI; scale bar, 10 μm. n = 3 mice, done in triplicate.

Figure 6.

Peak-to-peak amplitudes of ABR waves are not reduced in BDNF^Pax2 KO mice following noise exposure. ABR waves illustrate the difference in signal amplitude in control (A) and BDNF^Pax2 KO mice (B) before and 7 d after trauma (AT; ±SEM), shown for stimulation with clicks presented at 20 dB above the hearing threshold. C, Peak to peak amplitudes at 20 dB above hearing threshold for five selected peak-to-peak amplitudes (wave I-V, arrows in D, two-way ANOVA: p < 0.001). After exposure the amplitude of control mice was significantly reduced for waves II-V compared with pre-exposure (two-way ANOVA: p < 0.05). Amplitudes of BDNF^Pax2 KO mice were not significantly decreased after exposure. Control, n = 8/16 mice/ears; BDNF^Pax2 KO, n = 8/16.