Impaired auditory-vestibular functions and behavioral abnormalities of Slitrk6-deficient mice

Abstract

A recent study revealed that Slitrk6, a transmembrane protein containing a leucine-rich repeat domain, has a critical role in the development of the inner ear neural circuit. However, it is still unknown how the absence of Slitrk6 affects auditory and vestibular functions. In addition, the role of Slitrk6 in regions of the central nervous system, including the dorsal thalamus, has not been addressed. To understand the physiological role of Slitrk6, Slitrk6-knockout (KO) mice were subjected to systematic behavioral analyses including auditory and vestibular function tests. Compared to wild-type mice, the auditory brainstem response (ABR) of Slitrk6-KO mice indicated a mid-frequency range (8-16 kHz) hearing loss and reduction of the first ABR wave. The auditory startle response was also reduced. A vestibulo-ocular reflex (VOR) test showed decreased vertical (head movement-induced) VOR gains and normal horizontal VOR. In an open field test, locomotor activity was reduced; the tendency to be in the center region was increased, but only in the first 5 min of the test, indicating altered adaptive responses to a novel environment. Altered adaptive responses were also found in a hole-board test in which head-dip behavior was increased and advanced. Aside from these abnormalities, no clear abnormalities were noted in the mood, anxiety, learning, spatial memory, or fear memory-related behavioral tests. These results indicate that the Slitrk6-KO mouse can serve as a model of hereditary sensorineural deafness. Furthermore, the altered responses of Slitrk6-KO mice to the novel environment suggest a role of Slitrk6 in some cognitive functions.

Figure 1. Auditory function abnormalities in Slitrk6-KO mice.

(A) Representative waves of auditory brainstem response (ABR) from WT (left, n = 7) and Slitrk6-KO (right, n = 9) mice. ABR were recorded upon 0- to 80-dB sound pressure level (SPL) stimuli of 2, 4, 8, 16, and 24 kHz. The dash lines indicate the time of click presentation. The scale bars are shown at bottom right. (B) ABR thresholds in WT and Slitrk6-KO mice. Slitrk6-KO mice showed significantly higher thresholds to the 8- and 16-kHz stimuli than those of WT mice. (C) Top: Each peak number in a representative ABR wave. Middle: Comparison of the values of peaks I, II, and III between WT and Slitrk6-KO mice. Peak I of Slitrk6-KO mice was significantly reduced in the range of 8 to 16 kHz, and peak III was also significantly reduced at 24 kHz. Bottom: The latency to peaks I, II, and III. The latency did not show clear differences between WT and Slitrk6-KO mice. (D) Auditory startle response of WT (n = 10) and Slitrk6-KO mice (n = 10). Slitrk6-KO mice showed significantly lower startle responses to 95- to 120-dB sounds. *P<0.05, **P<0.01. All bars are mean ± standard error of the mean.

Figure 2. Vestibular function anomaly in Slitrk6-KO mice.

(A) Dynamic characteristics of horizontal optokinetic response (hOKR) of WT (n = 9) and Slitrk6-KO (n = 9) mice. (B) Horizontal vestibulo-ocular reflex (hVOR). There were no differences in gains (left) or phases (right) between WT (n = 9) and KO (n = 9) in hVOR or hOKR. (C) Vertical vestibulo-ocular reflex (vVOR) of WT (n = 8) and Slitrk6-KO mice (n = 6). Gains of vVOR (left) of the Slitrk6-KO mice were significantly smaller than those of WT. (D) Rota- rod test. Rotation indicates the speed of rotation (rpm) at which the mice fell off or revolved around the rod. The values were comparable between WT (n = 12) and Slitrk6-KO (n = 8) mice, suggesting that there were no strong deficits of balancing function in Slitrk6-KO mice. Values are mean ± standard error of the mean.

Figure 3. Spontaneous activity in home cage.

Each vertical bar represents standard error of the mean. ^# P = 0.068; *P<0.05. WT, n = 10; KO, n = 10. Below the graph, open and black bars indicate light phase (08:00–20:00) and dark phase (20:00–08:00), respectively.

Figure 4. Altered adaptive responses to a novel environment in Slitrk6-KO mice.

(A, B) Open field test. (A) The total distance moved was less in Slitrk6-KO than in WT mice (left). The distance moved in the first 5 min of the test was significantly lower in Slitrk6-KO mice (right). (B) In Slitrk6-KO mice, the total time spent in the center area was greater than that of the WT (left) and was significantly increased in the first 5 min of the test (right). (C–G) Hole-board test. There were no significant differences in distance moved (C), number of rearing episodes (D), and number of head dips (E) between Slitrk6-KO and WT mice. Duration per head dip was significantly decreased in Slitrk6-KO mice (F). Latency time to head dip was significantly decreased in KO mice (G). Values are mean ± standard error of the mean. *P<0.05, **P<0.01 for Mann-Whitney's U-test (B, center area data) or Student's t-test (others). WT, n = 10; KO, n = 10.

Figure 5. Normal anxiety level of Slitrk6-KO mice.