The authors have declared that no competing interests exist.
Conceived and designed the experiments: RN EM KO. Performed the experiments: RN. Analyzed the data: RN. Wrote the paper: RN EM KO.
Although some molecules have been identified as responsible for human language disorders, there is still little information about what molecular mechanisms establish the faculty of human language. Since mice, like songbirds, produce complex ultrasonic vocalizations for intraspecific communication in several social contexts, they can be good mammalian models for studying the molecular basis of human language. Having found that cadherins are involved in the vocal development of the Bengalese finch, a songbird, we expected cadherins to also be involved in mouse vocalizations.
To examine whether similar molecular mechanisms underlie the vocalizations of songbirds and mammals, we categorized behavioral deficits including vocalization in cadherin-6 knockout mice. Comparing the ultrasonic vocalizations of cadherin-6 knockout mice with those of wild-type controls, we found that the peak frequency and variations of syllables were differed between the mutant and wild–type mice in both pup-isolation and adult-courtship contexts. Vocalizations during male-male aggression behavior, in contrast, did not differ between mutant and wild–type mice. Open-field tests revealed differences in locomotors activity in both heterozygote and homozygote animals and no difference in anxiety behavior.
Our results suggest that cadherin-6 plays essential roles in locomotor activity and ultrasonic vocalization. These findings also support the idea that different species share some of the molecular mechanisms underlying vocal behavior.
The ability to speak and understand language is one of the most intellectual faculties of human beings. Although only humans are able to use language, components of language are seen in some nonhuman animals
Mice produce ultrasonic successive vocalizations in social contexts as pup’s isolation calls and courtship calls
In our previous study using a songbird, the Bengalese finch, we found (1) that cadherin-6B (the avian ortholog of the mammalian cadherin-6) and -7 are expressed in vocal control areas and the expression of cadherin-7 in the robust nucleus of the arcopallium (RA) is downregulated during the sensorimotor learning stage
(a–c) Spectrograms (frequency, kHz * time, s) of isolation calls produced by each genotype. (A,B) Mean and maximum (max) peak frequency in the Cad6−/− group were much higher (>75 kHz) than in the Cad6+/− and wild-type groups (<75 kHz). (C) The number of calls by Cad6−/− pups was larger than that by either the Cad6+/− or wild-type pups. (D) The latency to start calling in the Cad6−/− group was shorter than that in the Cad6+/− group, and there was no significant difference between the Cad6−/− and wild-type groups. Error bars represent the SEM.
(a–c) Spectrograms (frequency, kHz * time, s) of courtship songs produced by each genotype. (A) Mean peak frequency during 3 min was higher for the in Cad6−/− group (>75 kHz) than the wild-type group (<75 kHz). (B) Maximum (max) peak frequency was higher for the Cad6−/− group than either the Cad6+/− or wild-type group. (C) The number of calls during 3 min did not differ significantly in the 3 groups. (D) The latency to start calling did not differ significantly in the 3 groups. Error bars represent the SEM.
Basic sound features, mean and max peak frequency, # of calls, latency to start calling in both pup’s isolation calls (
Probability of ultrasonic calls in each of the 10 different call categories in (A) pup’s isolation calls and in (B) male’s courtship calls. Cad6−/− pups emitted more “Downward” calls and “short” calls, and fewer “More jumps” calls than WT pups (A). Cad6−/− males produced few “Harmonics” calls (p<.05) than WT, and there are also differences between Cad6+/− and WT males in “Flat” calls (p<.05) and “More jumps” calls (p<.01). Error bars represent the SEM.
(a–c) Spectrograms (frequency, kHz * time, s) of audible vocalizations produced by each genotype. (A) The peak frequency of audible vocalizations did not differ significantly between genotypes. Error bars represent the SEM.
(A) Running speed and (B) total distance were less than wild-type controls, but (C) the time spent in center did not differ significantly between genotypes. Error bars represent the SEM. (B),(C) Each data point represents the mean value in a one minutes bin.
To investigate the quantitative deficits in Cad6KO mice, we categorized each syllable as 1 of 10 distinct categories: “Cheveron”, “Complex”, “Downward”, “Flat”, “Short”, “Upward”, “Wave”, “Harmonics”, “One jump”, “More jumps”. Syllable category differences in each genotype are shown in
In the pup’s isolation call, the pattern of call category differed across call category (
These results thus suggest that Cad6 knockout mice have defects extending the frequency range and control the vocal repertoire of both the ultrasonic courtship song and the ultrasonic isolation call.
Exploring the possibility of defects in vocalizations produced by Cad6KO mice in the aggression call test, we found that the peak frequency of vocalizations in aggression behavior is not differed between groups [
To investigate the possibility that the deficits of the vocalization related to the abnormality of locomotor activity or anxiety levels, we used the open-field test to examine the animals’ amounts of locomotor activity and their presumptive anxiety levels. Cad6−/− mice run more slowly than did Cad6+/− and WT mice [
Analysis of the vocal behavior of Cad6KO mice revealed that both juvenile and adult homozygous mutant mice produced vocalizations with a higher pitch and unusual repertoire than did heterozygous and wild-type mice in both pup’s isolation calls and adult male’s courtship calls, but that vocalizations in male-male aggression behavior did not differ in these three groups. These results suggest that, as for vocalization behaviors, Cad6KO mice have defects only in the ultrasonic successive vocalizations, and that the defects are not caused due to impairment of peripheral vocal organs because they could vocalize in different social context. In addition, Cad6KO adult male mice showed deficits in the acoustic features and repertoire of calls but not the latency of vocalizations. This suggest that mechanisms controlling acoustic structures may be independent of the mechanisms controlling their motivation like how often and in which context do mice vocalize.
Since Cad6 is expressed in many brain areas of postnatal mouse brain–including the somatosensory cortex, motor cortex, and limbic system (
Many genes responsible for human language impairment–such as Robo1, KIAA0391, DCD2, and Dyx1C1–have been identified by linkage analysis of human patients
In this study, we found by analyzing Cad6KO mice that Cad6 is essential for proper ultrasonic vocalization. Many studies have shown that type-II cadherins (e.g., Cad6) are localized in the synapse and involved in synapse formation and function
Recently several researches proposed possibilities that mice ultrasonic vocalizations are basically innate
Cad6KO mice
The vocal behavior of Cad6KO mice was assessed by examining both ultrasonic and audible vocalizations, and their locomotor activity was assessed by open-field testing.
Vocalizations were examined under three conditions: (1) pup isolation (2) male courtship, and (3) male-male aggression context.
Fifty-one mice [26 Cad6−/−, 14 Cad6+/−, 11 wild type (WT)] were used on postnatal day 7. After each pup was removed from its huddling littermates and put into a 500-mL plastic beaker placed in a soundproof box, its vocalizations were recorded for 3 min. To maintain the pup’s body temperature, absorbent cotton was placed in the beaker. Condenser microphones (CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) 10 cm above the animal were connected, through a pre-amplifier (Avisoft Ultrasound Gate 416H; Avisoft Bioacoustics, Berlin, Germany), to a personal computer. Signals were recorded to the hard disk via Avisoft-Recorder USGH (Avisoft Bioacoustics, Berlin, Germany) set at a 300-kHz sampling rate, and the recorded sound was stored as “.wav” files.
Twenty-six mice (8 Cad6−/−, 8 Cad6+/−, and 10 WT) 13–17 weeks old were tested. Five WT female mice were used as stimulus animals. The stimulus mice were surgically ovariectomized seven days before the test, and estrogen was administrated chronically via a silastic tube. In each test trial the experimental male was placed in a plastic cage 30 s before a randomly selected stimulus female was put into the cage and vocalizations were recorded for 3 min using the same equipment used in the pup-isolation test.
Non-successive vocalizations in the lower pitch as the human audible range were also examined in a male-male aggression behavior test. 24 weeks old 19 animals (5 Cad6−/−, 7Cad6+/−, and 7 WT) are used as experimental subjects, and 5 WT mice used as intruders. Five weeks before the test the experimental animals and stimulus animals (i.e., intruders) were isolated in the breeding cages. Three days before the test the pharyngeal nerves of the stimulus animals were surgically extirpated. The audible vocalization test was performed in a plastic cage with a condenser microphone located 30 cm above and centered over the floor of the soundproof box. In each trial the experimental animal was put into the test cage 30 s before the stimulus animal was and recording then proceeded for 5 min.
The open-field test is commonly used to determine general activity levels, gross locomotor activity, and exploration habits in mice. We used it to examine whether the Cad6 knockout animals show abnormal behavior as measured by the amount of activity and emotional behavior. Thirty-two 8-week-old mice (12 Cad6−/−, 9 Cad6+/−, and 11 WT) were tested. Each animal was placed in the center of the open-field box (50 cm×50 cm×40 cm high) and allowed to move freely for 10 min while being tracked by a system using ImageJ software. The floor of the box was separated into center and corner areas by virtual lines making a 5*5 grid, and in each 10-min trial the total distance traveled, mean running speed, and time spent in the center (10 cm from the wall) were recorded. The floor of the open-field box was cleaned with 70% ethanol after every trial.
The recorded files were transferred to SASLab Pro (ver. 4.52, Avisoft Bioacoustics, Berlin, Germany) for fast Fourier transform analysis (FFT length 512, 100% frame size, 100% frame size, Hamming window, 50% time window overlap) with a 20-kHz high-pass filter. In both the isolation and courtship contexts we analyzed the number of syllables, the latency to start calling, and the mean peak frequency of each syllable. In the audible vocalization test we analyzed only mean peak frequency after background noise was reduced by the GoldWave program.
Waveform pattern of syllables were analyzed in the sonograms collected from every genotype (pup’s isolation call: 531 WT syllables, 1188 Cad6+/− syllables; 1724 Cad6−/− syllables; adult male’s courtship call: 1931 WT syllables, 408 Cad6+/− syllables, 1777 Cad6−/− syllables). Each call is categorized as the 1 of 10 distinct categories, based on internal pitch change, length, and shape, according to previously reported categories with minor modifications
One-way or two-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) test was used for statistical analysis. Probability of vocalizations was standardized by angular transformation before analyzed.
We thank Dr. Masatoshi Takeichi for cadherin-6 KO mice.