Hearing function was investigated in male and female F344 rats in five age groups: 3-month-, 8-month-, 12-month-, 22–24-month- and 27–30-month-old animals. The animals in all of the age groups were in good health. HEINE mini 2000 otoscope was used to exclude any eardrum pathology. Fischer 344 rats were obtained from Charles River Deutschland (Sulzfeld, Germany) and subsequently maintained in a local facility through inhouse breeding. All of the animals were housed in age-matched groups of two or three per cage, under standard laboratory conditions, in a constant environment and a 12/12 h normal light/dark cycle; food and water were available ad libitum. All experimental procedures were approved by the Ethical Committee of the Institute of Experimental Medicine of the Czech Academy of Sciences, and followed the guidelines of the EU Directive 2010/63/EU for animal experiments.

ABRs were recorded in animals placed in a sound proof and anechoic room using acoustical free-field stimulation. The walls and ceiling inside the room were covered by cones from phono-absorbent material. Acoustic measurements demonstrated that the attenuation inside the room against the noise level outside the room, was 55 dB at 250 Hz and 60–70 dB for frequencies above 500 Hz. The acoustic system inside the room was calibrated with a Bruel&Kjaer^® 4939 microphone, a ZC0020 preamplifier and a B&K 2231 sound level meter (BRÜEL & KJÆR SOUND & VIBRATION MEASUREMENT A/S, Nærum, Denmark). The frequency–response curve of this system was relatively flat and varied by less than ±9 dB between 0.15 kHz and 40 kHz.

The hearing thresholds in rats were assessed by recording the ABRs in the animals under light anesthesia (ketamine 35 mg/kg, xylazine 6 mg/kg), using subcutaneous needle electrodes placed on the vertex (an active electrode) and in the neck muscles (ground and reference electrodes). The signal from the electrode was amplified 10,000-times using RA16PA RA4LI preamplifier (Tucker Davis Technologies, SystemIII, Alachua, FL, USA), band-pass filtered over the range of frequencies from 300 Hz to 3 kHz, processed with a TDT RX5-2 Pentusa Base Station (Tucker Davis Technologies, SystemIII, Alachua, FL, USA) and analyzed with BioSig software.

ABRs were evoked by tonal stimuli (3-ms duration, 1 ms rise/fall times) in the frequency range from 2 kHz to 40 kHz, or by clicks generated with a RP2.1 enhanced real-time processor (Tucker Davis Technologies, SystemIII, Alachua, FL, USA). To test the neural adaptability, a series of four clicks with variable inter-click interval (ICI) ranging from 1 ms to 50 ms were used. Acoustic stimuli were delivered in free-field conditions via a two-way loudspeaker system [Jamo^® 22496 woofer (Jamo Denmark ApS, Niva, Denmark) and SEAS Excel^® T25CF 002 tweeter (SEAS FABRIKKER AS, Moss, Norway)], placed 70 cm in front of the animal’s head. The ABR threshold to each frequency was determined as the minimal tone intensity that still evoked a noticeable potential peak in the expected time window of the recorded signal.

To test the functional status of the OHC, the DPOAEs were recorded with a low-noise microphone system (Etymotic probe ER-10B+; Etymotic Research, Elk Grove Village, IL, USA). DP-grams (the function of the DPOAE level on increasing stimulus frequency) were recorded with a resolution of four points per octave over the frequency range from 1 kHz to 38 kHz. During DPOAE recording, in addition to DPOAE amplitude, also the F1 and F2 intensities in the outer ear canal were monitored to confirm the correct placement of the recording probe. Acoustic stimuli (two primary tones with frequency ratio F₂/F₁ = 1.21 and level L₁ = L₂ = 65 dB) were generated by a TDT System III (RP2 processor; sampling rate 100 kHz) and presented to the ear canal with two custom-made piezoelectric stimulators connected to the probe with 10-cm-long silastic tubes. The signal from the microphone was analyzed by a TDT System III (RP2 processor; sampling rate 100 kHz). DPOAEs were measured in both ears of the animals. The recording of DPOAEs were performed in an anechoic and sound proof room.

During ABR and DPOAE recordings, the rats were placed on a heating pad that automatically maintained body temperature at 38°C.

Deeply anesthetized animals (ketamine 55 mg/kg and xylazine 8 mg/kg i.m.) were sacrificed and cochleas were quickly removed from the skull. Holes were made in the apical turn, round and oval windows, and cochleas were immersed in 4% paraformaldehyde solution for 3 h (whole mount preparation, n = 4 per group) or overnight (paraffin sectioning, n = 8 in young groups, n = 20 in old groups). One ear from each animal was used for cochlear cross-section preparation; the opposite ear was used for cochlear whole mounts in some animals.

For whole mount preparation the bone of the cochlear wall and tectorial membrane were removed gradually from the cochlear apex to the base and basilar membrain together with organ of Corti was microdissected into four pieces per each cochlea. Cochlear whole mounts then were stained with anti-C-terminal binding protein 2 antibodies (anti-CtBP2, BDTransduction Laboratories, San Jose, CA, USA) for presynaptic hair cell ribbons analysis, phalloidin (# 59033, Dyomics) for hair cell visualization, and DAPI to counterstain cell nuclei. Cochlear preparations were visualized using confocal microscope Zeiss LSM 510 Duo. For each analyzed cochlea, low magnification images of all of the whole mount parts were acquired and localization of certain frequency was determined, based on a percentage distance from base using ImageJ Plugin Measure_Line¹.

The numbers of OHCs and IHC ribbons were counted using ImageJ software (Schneider et al., 2012). Part of the basilar membrane, hosting 60 OHCs (20 in each raw) in the proximity of certain frequency was analyzed. Present and missing cells were counted and the percentage of cells present was calculated. The mean value for each frequency from four cochleas per group was plotted in the resulting graph. IHC ribbon synapses were counted in image stacks. Five adjacent IHC in the proximity of the particular frequency were chosen and the synaptic puncta relating to them were counted, using Cell counter plugin of ImageJ software (Schneider et al., 2012). The number of synaptic puncta per one IHC was calculated for each frequency in each cochlea.

To analyze the SV, cochlear cross-sections were used. The fixed tissues were decalcified in ethylendiaminetetraacetic acid (0.5 M, pH 8.0) for 2 weeks, dehydrated through a graded alcohol series, cleared in toluene, and embedded in parafin. The paraffin blocks were cut into 5-μm horizontal serial sections. The slices were deparaffinized and hydrated. Tissue sections were treated with Bouin’s solution to enhance the final coloration and stained with Weigert’s iron hematoxylin and Masson’s trichrome staining kit (HT15-1KT, Sigma-Aldrich) visualizing collagen. The changes of stria vascularis morphology were analyzed using light microscope Leica DMRXA.

The differences between ABR hearing thresholds, DPOAE amplitudes, number of OHCs and IHC ribbon synapses in F344 males and females were tested using two-way ANOVA and Bonferroni post hoc test. The differences between ABR amplitudes and ABR thresholds at 8 kHz tones were tested using an unpaired t-test.

The average ABR hearing thresholds (ABR audiograms) for F344 male and female rats of different ages are shown in Figure 1. The ABR thresholds in young male and female F344 rats (3-months-old) were found to be almost identical (Figure 1A, n = 10m + 10f). In the higher age categories the ABR thresholds increased, but the hearing alterations started later in the F344 females and progressed more slowly than in the F344 males. In contrast, the ABR thresholds in females aged 8 months (Figure 1B, n = 4m + 4f) were the same as in 3-month-old females, while the ABR thresholds in 8-month-old males increased in comparison with 3-month-old males, and they were significantly higher at 32–40 kHz in comparison with 8-month-old females (p < 0.05, Bonferroni post hoc test). In 12-month-old males (Figure 1C, n = 6m + 6f) the ABR thresholds were significantly higher, in comparison with age-matched females at frequencies 4–32 kHz (p < 0.05 and p < 0.001, Bonferroni post hoc test). In 22–24-month-old males (Figure 1D, n = 8m + 6f), the ABR thresholds were significantly higher at frequencies 2–16 kHz in comparison with females (p < 0.05 and p < 0.001, Bonferroni post hoc test). In 27–30-month-old males (Figure 1E, n = 10m + 8f), the hearing loss increased more rapidly in females than in males which resulted in a decrease of differences between hearing thresholds in these oldest females and males. Even though the ABR thresholds in F344 males and females were significantly different (p < 0.0001, two-way ANOVA), the differences in individual frequencies was significant only at frequency 40 kHz (p < 0.05, Bonferroni post hoc test).

Figure 1F demonstrates the comparison of the time pattern of ABR threshold changes at 8 kHz for males and females. In males the ABR threshold at 8 kHz increased continuously, and almost linearly, from the age of 3 months to the age of 27–30 months. In females this threshold increase started later (in females older than 8 months) and continued to the age category 22–24 months. However, the ABR threshold at 8 kHz in the F344 females aged 22–24 months started to increase with a faster rate than in males and at the age of 27–30 months, ABR thresholds in females almost reached the thresholds in the age-matched F344 males. Whereas ABR thresholds at 8 kHz in 22–24 months old males and females were significantly different (p < 0.0023, unpaired t-test), ABR thresholds at 8 kHz in 27–30 month old males and females were not significantly different (p < 0.114, unpaired t-test; Figure 1F).

Figure 2 shows age-related changes in the ABR amplitudes, evoked by clicks at 100 dB SPL, and measured as the difference between the value of the positive peak II and the negative through between the peaks II and IV (see inserted schema). The results indicate that ABR amplitudes are significantly smaller in F344 males aged 8–24 months than in age-matched females (p < 0.05 to p < 0.001; unpaired t-test).

Amplitudes of individual ABR waves, evoked by click stimulation of variable intensity, were measured in five males and five females of 24-month old F344 rats. The averaged amplitude-intensity functions for the ABR wave I, II and IV are demonstrated in Figure 3. Maximal wave I amplitude (Figure 3A) in females was three times the maximal wave I amplitude in males. The difference in maximal wave amplitudes is less expressed in later ABR waves. The maximal wave II amplitude (Figure 3B) in females is twice that of males and the maximal wave IV amplitude (Figure 3C) is only 1.6 times that in males. These results indicate that the difference between ABR wave I amplitude, originating at the periphery, in males and females, is larger than the difference between ABR wave IV amplitude, originating in higher auditory structures.

To test the neural adaptation in five males and five females of 24-month old F344 rats, stimulation with a series of four clicks, intensity 70 dB SPL, with variable interclick interval (ICI) was used. Figure 4A demonstrates the schema of an experiment. When the ICI was short (ICI = 3 ms), ABR amplitudes to 2nd, 3rd and 4th clicks were smaller than ABR amplitude to 1st click stimulation. When the ICI was longer (ICI = 5 ms), ABR amplitudes to 2nd, 3rd and 4th click were the same as ABR amplitudes to 1st click stimulation. The function of ABR amplitudes on ICI intervals in aged males and females is demonstrated in Figure 4B. As the ABR amplitudes to the 2nd, 3rd and 4th click stimulation within a particular click series were similar, Figure 4B compares the ABR amplitudes to the 1st and 2nd click stimulation in the F344 males and females. Regardless of the smaller click-evoked ABR amplitude obtained in males, the function of the ABR amplitude on ICI was similar in both sexes, i.e., the amplitude of ABR evoked by the second click, reached the ABR amplitude of the first click at ICI of 4 ms.

The functional status of cochlear OHC were checked by the recording of DPOAEs. The average DP grams (i.e., the function of DPOAE amplitudes above background noise level on F2 stimulus frequency) in six to eight F344 males and females of different age category are present in Figure 5. The DPOAE amplitudes of both males and females is maximal in young, 3-month-old animals (Figure 5A). At a higher age the DPOAE amplitudes of both males and females decreased. In animals aged 12 months (Figure 5C), the DPOAE amplitudes in F344 males were significantly smaller than those in F344 females (p < 0.0001, two-way ANOVA), but Bonferroni post hoc test did not show any significant difference at individual F2 frequencies. In males older than 20 months, the DPOAEs were practically absent, whereas in some of the 22–24-month-old females, the DPOAEs were still measurable (Figure 5D). In the age category 27–30 months no DPOAE were detectable in either male or female F344 rats (not presented).

Cochlear whole mount preparations were used to calculate the number of OHCs and the number of ribbon synapses docking vesicles with neurotransmitter glutamate in the IHCs in 27-month-old F344 males (n = 4) and females (n = 4). In Figure 6, examples of cochlear whole mounts from aged rats, showing IHC ribbon synapses immunostained with CtBP2 protein (green), in males (Figure 6A) and females (Figure 6B), and IHCs and OHCs stained with phalloidin (red) in males (Figure 6C) and females (Figure 6D), are presented. The average number of surviving OHCs in individual tonotopical regions of the organ of Corti are presented in Figure 6E. The pronounced OHC loss was observed mainly at the apical, low-frequency end of the cochlea (50%–60%), whereas only a few OHCs were missing at the basal, high-frequency part of the cochlea (less than 10%). However, the difference between both curves is not statistically significant (p = 0.364; two-way ANOVA). The quantitative analysis of the number of IHC ribbon synapses per one IHC (Figure 6F) demonstrated that the average number of ribbon synapses per IHC is almost identical in both F344 males and females (p = 0.071; two-way ANOVA).