Engineered deafness reveals that mouse courtship vocalizations do not require auditory experience

Abstract

Auditory experience during development is necessary for normal language acquisition in humans. Although songbirds, some cetaceans, and maybe bats may also be vocal learners, vocal learning has yet to be well established for a laboratory mammal. Mice are potentially an excellent model organism for studying mechanisms underlying vocal communication. Mice vocalize in different social contexts, yet whether they learn their vocalizations remains unresolved. To address this question, we compared ultrasonic courtship vocalizations emitted by chronically deaf and normal hearing adult male mice. We deafened CBA/CaJ male mice, engineered to express diphtheria toxin (DT) receptors in hair cells, by systemic injection of DT at postnatal day 2 (P2). By P9, almost all inner hair cells were absent and by P16 all inner and outer hair cells were absent in DTR mice. These mice did not show any auditory brainstem responses as adults. Wild-type littermates, also treated with DT at P2, had normal hair cells and normal auditory brainstem responses. We compared the temporal structure of vocalization bouts, the types of vocalizations, the patterns of syllables, and the acoustic features of each syllable type emitted by hearing and deaf males in the presence of a female. We found that almost all of the vocalization features we examined were similar in hearing and deaf animals. These findings indicate that mice do not need auditory experience during development to produce normal ultrasonic vocalizations in adulthood. We conclude that mouse courtship vocalizations are not acquired through auditory feedback-dependent learning.

Figure 1.

Experimental timeline. Injection of DT was given at P2. The majority of inner hair cells were gone by P9, and all cochlear hair cells were eliminated by P16 in Pou4f3^+/DTR mice. *Auditory brainstem responses obtained.

Figure 2.

Low- and high-power confocal images of whole-mount preparations from representative P9 mice. A–C, WT (Pou4f3^+/+) mouse. D–F, Pou4f3^+/DTR mouse. Both mice were injected with DT on P2. Red cells show antigenicity to SOX2, indicating an organ of Corti support cell phenotype; hair cells are indicated by antigenicity to a mixture of antibodies against myosin6 and parvalbumin, and blue represents DAPI-stained nuclei. A, In WT mice, the low-power image shows that the full complement of hair cells was present throughout the basal to apical turns. B, High-power image shows the characteristic single row of inner hair cells, a space for the tunnel of Corti, and then three rows of outer hair cell somata in the basal region. C, Middle turn region. D, In the Pou4f3^+/DTR mice, however, the low-power image reveals a dramatic loss of hair cells by 7 d after the DT injection. E, High-power images reveal complete loss of inner hair cells and almost complete loss of outer hair cells in the basal region, coupled with what appears to be complete survival of supporting cells. F, In the middle region, an occasional inner hair cell remained, but it usually appeared swollen and degenerative, whereas a large complement of outer hair cells had not yet degenerated. Again, supporting cell complement appeared intact. By P16, all hair cells had been lost (data not shown). Scale bars: (in A) A, D, 100 μm; (in B) B, C, E, F, 10 μm.

Figure 3.

Pou4f3^+/DTR mice were deaf. ABRs from all male mice were obtained at P70. Thresholds are mean ± SD for hearing (♦) and deaf (×) animals. The highest level tested with the DTR mice was 90 dB SPL, and none of the animals had any response at this intensity.

Figure 4.

Vocalizations of hearing and deaf male mice were qualitatively similar. A, Example spectrograms of ultrasonic vocalizations emitted by one hearing and one deaf male mouse in the presence of a female. B, Mean number of syllables emitted per minute >15 min recording sessions of hearing and deaf mice. The circles represent the mean values for each animal, and the bars represent the mean values for the group. Error bars indicate the SDs of the group means.

Figure 5.

The temporal organization of vocalizations emitted by hearing and deaf mice was similar. A, Fraction of syllables contained in bouts. B, Number of bouts emitted per minute. C, Number of syllables per bout. D, Intersyllable interval. In all plots, the circles represent the mean values for each animal, and the bars represent the mean values for the group. Error bars indicate the SDs of the group means.

Figure 6.

Hearing and deaf mice emitted the same types of syllables. The relative occurrence of each syllable type for hearing and deaf animals. For each syllable, the circles represent the mean values for each animal, and the bars represent the mean values for the group. Error bars indicate the SDs of the group means.

Figure 7.

The pattern of syllable emissions was not different between hearing and deaf males. Transition probability matrices for two hearing males, two deaf males, and the hearing and deaf group means. Syllable categories 1–12 are as follows: one jump, two jump, three jump, four jump, five jump, FM upsweep, FM downsweep, reverse chevron, chevron, constant frequency, complex, and short, respectively. S, Silence.

Figure 8.

Acoustic parameters in jump syllables were not different for hearing and deaf animals. In all plots, the circles represent the mean values for each animal, and the bars represent the mean values for the group. Error bars indicate the SDs of the group means.

Figure 9.

Acoustic parameters in nonjump syllables were not different for hearing and deaf animals. In all plots, the circles represent the mean values for each animal, and the bars represent the mean values for the group. Error bars indicate the SDs of the group means.

Figure 10.

The variability of vocalization parameters within an animal was high. A, Histogram of syllables per bout for all of one animal's vocalizations. B, Syllables per bout for each animal (mean ± SD) and group mean ± SD.