- Email: [email protected]
Severe hearing loss in Dlx1 mutant mice
Hearing Research 214 (2006) 84–88
Hearing Research
Research paper
Severe hearing loss in Dlx1 mutant mice Daniel B. Polley
a,1
, Inma Cobos b, Michael M. Merzenich a, John L.R. Rubenstein
b,*
a
b
Coleman Memorial Laboratory, W.M. Keck Foundation Center for Integrative Neuroscience, Department of Otolaryngology, University of California, San Francisco, CA 94143, USA Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, CA 94158, USA Received 15 July 2005; received in revised form 16 February 2006; accepted 16 February 2006
Abstract The Dlx homeobox gene family participates in regulating middle and inner ear development. A significant role for Dlx1, in particular, has been demonstrated in the development of the middle ear ossicles, but the functional consequences of Dlx1 gene mutation on hearing thresholds has not been assessed. The present study characterizes auditory brainstem responses to click and tonal stimuli in a non-lethal variant of a Dlx1 gene knockout. We found that peripheral hearing thresholds for click and tonal stimuli were significantly elevated in homozygous Dlx1 knockout (Dlx1 / ) compared to both heterozygous (Dlx1+/ ) and wild type (Dlx1+/+) mice. Thus, abnormal morphogenesis of the incus and stapes that has been documented previously with histological measures is now known to result in a severe peripheral hearing deficit. Ó 2006 Elsevier B.V. All rights reserved. Keywords: ABR; Branchial arch; Incus; Stapes; Mouse; Ossicles; Development
1. Introduction Dlx1, 2, 3, 4, 5 & 6 genes encode homeodomain transcription factors that have essential functions in multiple regions of prenatal and postnatal mammals. The mouse Dlx genes are expressed in multiple tissues including the embryonic brain, face and limbs (Depew et al., 1999; Depew et al., 2002; Ellies et al., 1997; Ferrari and Kosher, 2002; Panganiban and Rubenstein, 2002; Qiu et al., 1997). In addition to regulating early limb morphogenesis, Dlx genes regulate patterning and differentiation of skeletal tissues (Acampora et al., 1999; Beverdam et al., 2002; Depew et al., 1999; Depew et al., 2002; Ferrari and Kosher, 2002; Abbreviations: ABR, auditory brainstem response; PCR, polymerase chain reaction; dB SPL, decibels sound pressure level; kHz, kilohertz * Corresponding author. E-mail addresses: [email protected] (D.B. Polley), john. [email protected] (J.L.R. Rubenstein). 1 Present address: Department of Hearing and Speech Sciences, Vanderbilt University, 465 21st Ave. South, 7110 MRB III, Nashville, TN 37232-8548, USA. Tel.: +615 343 0577; fax: +615 936 3745. 0378-5955/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.02.008
Harris et al., 2003; Hassan et al., 2004; Lezot et al., 2002; Thomas et al., 1997). Within the otic vesicle, different Dlx genes are expressed in distinct patterns that correlate with its functional subdivisions. Dlx5 expression is detected in the lagena macula and the cochlear and vestibular nerves. Dlx1 and Dlx2 expression, in contrast, have not been detected in the otic vesicle; but are found in non-neuronal cells of the cochleovestibular ganglion and nerves (Brown et al., 2005). Although Dlx1 has been known to regulate development of the middle ear for some time (Qiu et al., 1997), the specific consequences of Dlx1 mutations on middle ear morphogenesis has only been described recently (Depew et al., 2005). The more detailed analysis of middle ear morphology in Dlx1 / mice performed in this recent study revealed that the caudal processus brevis and processus longus of the incus can develop as unattached to the rest of the incus. In approximately 50% of the cases, the stapes is hypoplastic and lacks a foramen (through which the stapedial artery normally traverses); in the cases when the foramen forms, it is usually asymmetrically placed. It was
D.B. Polley et al. / Hearing Research 214 (2006) 84–88
also shown that the effects of Dlx1 mutation are titrated according to the number of functional alleles. Mice in the heterozygous state exhibited abnormalities in the basal lamina of the ala temporalis in a similar fashion to the homozygous mice, but the formation of the ossicles appeared normal. The present study seeks to expand our understanding of Dlx1 gene mutations on middle ear development by providing a functional analysis of hearing thresholds to compliment previous histological analyses.
85
acteristic of that seen at higher intensities. In some cases, a response could not be detected at the highest intensity tested. These cases are marked separately in Fig. 2A–E. For the purposes of statistical comparisons, however, these cases were scored as having a response threshold equal to 70 dB SPL. Thresholds were compared for each stimulus type with a Mann–Whitney U statistic. All recordings and analyses were performed blind with respect to genotype.
2. Materials and methods Dlx1 / mice used in this study were initially made using 129-strain ES cells. The mice were initially carried on the C57BL/6J strain, and then out-crossed for at least 5 generations onto the CD1 strain to increase their viability. PCR genotyping followed the methods described in Qiu et al. (1997). Fifteen mice (N = 5 for Dlx1 / , Dlx1+/ , and Dlx1+/+groups) of the C57BL/6J;CD1 hybrid strain were used in this study. All mice were 7–8 weeks at the time of recording. Prior to electrode placement, animals were anesthetized with ketamine (100 mg/ kg i.p.) and medetomidine (0.3 mg/kg i.p.). Body temperature was maintained near 37.5 °C with a rectal probe and homeothermic blanket (Harvard Instruments). Auditory brainstem response (ABR) measurements were performed in a sound-attenuated chamber (Acoustic Systems). ABRs were recorded with silver wire electrodes (0.13 mm diameter, A–M systems) threaded through the skin at three locations: The positive electrode was placed directly behind the right ear over the bulla, the negative electrode over the vertex, and the ground electrode was placed over the neck muscles. ABR signals were acquired, filtered, amplified, and analyzed with equipment and software manufactured by Tucker-Davis Technologies (Alachua, FL). Click and tone pip stimuli were presented with a freefield speaker (Vifa) positioned 22 cm from the external auditory opening. Tone pips (1.2 ms duration, 0.2 ms raised cosine ramps) were presented at 4, 8, 16 and 32 kHz. Acoustic calibration was performed with a microphone (Bruel & Kjaer) to ensure that tone and click stimuli were presented at the specified intensity. Auditory thresholds were obtained for click and tonal stimuli. Click thresholds were determined by presenting 500 click stimuli (10/s) at 50 dB SPL and reducing the sound level in 5 dB SPL steps until the response pattern was no longer visible. If no response was visible at 50 dB SPL, the sound level was increased in 5 dB SPL steps up to a maximum of 70 dB SPL. Tone thresholds were determined in a similar fashion with the exception that sound levels were changed in 10 dB SPL steps rather than 5 dB SPL steps and ABR traces were generated with 1000 stimulus presentations rather than 500. The entire range of sound levels was presented for one animal in each group to obtain a complete set of ABR records for illustrative purposes (Fig. 1). Auditory thresholds were defined for each stimulus as the lowest sound intensity capable of eliciting a response pattern char-
Fig. 1. Determination of ABR thresholds. Representative ABR patterns in a Dlx+/+ mouse (A), Dlx+/ mouse (B), and Dlx / mouse (C) elicited by a 8 kHz tone stimulus presented at sound levels ranging from 0–70 dB SPL. All traces are shown on the same fixed amplitude scale indicated by the scale bar in (A). Open arrows indicate the time at which the sound stimulus reaches the external auditory opening. Closed arrows indicate the threshold response for each mouse. Inset: the threshold response is presented on a normalized scale to illustrate the preservation of the response pattern despite the reduction in absolute amplitude.
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Fig. 2. Quantification of ABR thresholds. (A–E) Response thresholds measured with click and tonal stimuli. Each dot represents the threshold measurement obtained from a single mouse. (F) Mean ± standard error ABR thresholds for Dlx+/+mice (open circles), Dlx+/ mice (gray triangles), and Dlx / mice (black squares) for each stimulus type. Asterisks indicate significant differences (p < 0.025) between Dlx+/+and Dlx / groups based on the Mann–Whitney U statistic.
Confidence intervals were set according to the Bonferroni adjustment for multiple comparisons. 3. Results The ABR waveforms evoked by an 8 kHz tone pip are shown for one representative mouse from each group in Fig. 1. Auditory stimulus presentation evoked the characteristic multi-peaked waveform that became progressively weaker as sound level decreased. While the overall shape of the ABR is similar between groups, the 8 kHz response threshold was substantially elevated in Dlx1 / mice (Fig. 1C) compared to Dlx1+/ (Fig. 1B) and Dlx1+/+ (Fig. 1A) mice. We also observed elevated response thresholds in Dlx1 / mice for other types of auditory stimuli. Response thresholds for each stimulus type are shown in Fig. 2A–E for each individual mouse. Click-evoked response thresholds were 46 dB SPL higher in Dlx1 / mice than
Dlx1+/+ controls (mean ± standard error = 58.0 ± 2.6 dB SPL vs. 12.0 ± 2.0 dB; p < 0.01; Fig. 2A) yet click thresholds in Dlx1+/ mice were not significantly different than Dlx1+/+ controls (15.0 ± 2.04; p = 0.3). Note that a clickevoked threshold could not be calculated for one Dlx1+/ mouse because it died prematurely. Stimulus evokedresponses to 4 kHz tones were not observed at any sound level in any Dlx1 / mice. Accordingly, we observed a significant difference in 4 kHz response threshold between Dlx1+/+ and Dlx1 / mice (Dlx1+/+ = 40.0 ± 3.16 dB SPL; p < 0.005; Fig. 2B) but not between Dlx1+/+ and Dlx1+/ mice (Dlx1+/ mice = 44.0 ± 2.45 dB SPL; p = 0.34). Response thresholds for 8 kHz tones were 48 dB SPL higher on average in Dlx1 / mice compared to Dlx1+/+ controls (64.0 dB SPL ± 2.45 vs. 16.0 ± 2.45 dB; p < 0.01; Fig. 2C) but were not significantly different between Dlx1+/+ and Dlx1+/ mice (22.0 ± 3.74 dB; p = 0.21). Similarly, 16 kHz response thresholds in Dlx1 / mice were 32 dB SPL higher on
D.B. Polley et al. / Hearing Research 214 (2006) 84–88
average than Dlx1+/+ controls (48.0 ± 3.74 dB SPL vs. 16.0 ± 2.45 dB; p < 0.01; Fig. 2D) but were not different than Dlx1+/ mice (22.0 ± 3.74 dB; p = 0.21). Response thresholds at 32 kHz, in contrast, were not significantly different between Dlx1+/+mice (66.0 ± 4.0 dB) and Dlx1 / mice (66.0 ± 2.5 dB; p = 0.7) or Dlx1+/ mice (56.0 ± 6.0 dB; p = 0.19; Fig. 2E). 4. Discussion In summary, we have shown that Dlx / mice exhibit significantly elevated hearing thresholds for frequencies less than 32 kHz compared to Dlx1+/+ controls. The lack of a threshold difference at 32 kHz may be attributable to a diminished sensitivity to high-frequency sounds that is known to occur in C57BL/6J mice irrespective of the Dlx1 mutation rather than a partial sparing of hearing thresholds in Dlx / mice (Henry and Chole, 1980; Willott, 1986). Although the onset of age-related hearing loss in C57B6 mice does not typically occur until mice are several months older than those used in this study, we used C57B6/CD1 hybrid mice in which the exact progression of age-related high-frequency hearing loss is unknown. Hearing thresholds in Dlx1+/ mice, in contrast, were statistically indistinguishable from Dlx1+/+ controls although there was a trend for a slightly elevated thresholds for frequencies less than 32 kHz. Abnormal middle ear morphogenesis previously documented in homozygous Dlx1 mutants with histological methods has been shown in the present study to result in severe hearing loss. This hearing loss is likely to be exclusively attributable to middle ear dysfunction as there is no evidence for Dlx1 expression in the inner ear, nor any evidence for abnormal cochlear development in Dlx1 mutants (Depew et al., 2005; Qiu et al., 1997). In addition to their established role in craniofacial development, Dlx genes are also known to regulate various aspects of interneuron migration and survival in the forebrain (Panganiban and Rubenstein, 2002). Specifically, Dlx1 / mice have recently been shown to exhibit a selective loss of somatostatin+ and calretinin+ interneurons in the cerebral cortex and an associated increase in the incidence of generalized electrographic seizures (Cobos et al., 2005). The present study was motivated by an interest in characterizing abnormalities in physiological organization and stimulus processing in the auditory cortex of Dlx1 / mice. Clearly, we could not pursue these experiments given the severe peripheral hearing deficit observed in homozygous mice. Nevertheless, preliminary evidence suggests that the Dlx1+/ mice may also exhibit a cortical phenotype (JLRR, unpublished data). Given that these mice have relatively normal hearing thresholds, Dlx1+/ mice might prove to be a valuable model for future studies interesting in the link between Dlx1 gene expression and the physiological organization of central auditory nuclei.
87
Acknowledgements We thank Jacob Bollinger for technical assistance. This work was supported by the research grants to J.L.R.R. from Nina Ireland, Hillblom Foundation, March of Dimes, NIDCD R01 DC005667 and NIMH K05 MH065670; to I.C. from National Alliance for Research on Schizophrenia and Depression; to D.B.P. from NIH fellowship F32 DC005711; to M.M.M. from the Coleman Fund, The Sooy Fund, and NIH grants NS-10414 and NS-38416. References Acampora, D., Merlo, G.R., Paleari, L., Zerega, B., Postiglione, M.P., Mantero, S., Bober, E., Barbieri, O., Simeone, A., Levi, G., 1999. Craniofacial, vestibular and bone defects in mice lacking the Distalless-related gene Dlx5. Development 126, 3795–3809. Beverdam, A., Merlo, G.R., Paleari, L., Mantero, S., Genova, F., Barbieri, O., Janvier, P., Levi, G., 2002. Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: mirror of the past? Genesis 34, 221–227. Brown, S.T., Wang, J., Groves, A.K., 2005. Dlx gene expression during chick inner ear development. J. Comp. Neurol. 483, 48–65. Cobos, I., Calcagnotto, M.E., Vilaythong, A.J., Thwin, M.T., Noebels, J.L., Baraban, S.C., Rubenstein, J.L., 2005. Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat. Neurosci. 8, 1059–1068. Depew, M.J., Liu, J.K., Long, J.E., Presley, R., Meneses, J.J., Pedersen, R.A., Rubenstein, J.L., 1999. Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126, 3831– 3846. Depew, M.J., Lufkin, T., Rubenstein, J.L., 2002. Specification of jaw subdivisions by Dlx genes. Science 298, 381–385. Depew, M.J., Simpson, C.A., Morasso, M., Rubenstein, J.L., 2005. Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development. J. Anat. 207, 501–561. Ellies, D.L., Stock, D.W., Hatch, G., Giroux, G., Weiss, K.M., Ekker, M., 1997. Relationship between the genomic organization and the overlapping embryonic expression patterns of the zebrafish dlx genes. Genomics 45, 580–590. Ferrari, D., Kosher, R.A., 2002. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev. Biol. 252, 257–270. Harris, S.E., Guo, D., Harris, M.A., Krishnaswamy, A., Lichtler, A., 2003. Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors. Front Biosci. 8, s1249–s1265. Hassan, M.Q., Javed, A., Morasso, M.I., Karlin, J., Montecino, M., van Wijnen, A.J., Stein, G.S., Stein, J.L., Lian, J.B., 2004. Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol. Cell. Biol. 24, 9248–9261. Henry, K.R., Chole, R.A., 1980. Genotypic differences in behavioral, physiological and anatomical expressions of age-related hearing loss in the laboratory mouse. Audiology 19, 369–383. Lezot, F., Descroix, V., Mesbah, M., Hotton, D., Blin, C., Papagerakis, P., Mauro, N., Kato, S., MacDougall, M., Sharpe, P., Berdal, A., 2002. Cross-talk between Msx/Dlx homeobox genes and vitamin D during tooth mineralization. Connect. Tissue Res. 43, 509–514. Panganiban, G., Rubenstein, J.L., 2002. Developmental functions of the Distal-less/Dlx homeobox genes. Development 129, 4371–4386. Qiu, M., Bulfone, A., Ghattas, I., Meneses, J.J., Christensen, L., Sharpe, P.T., Presley, R., Pedersen, R.A., Rubenstein, J.L., 1997. Role of the Dlx homeobox genes in proximodistal patterning of the branchial
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arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev. Biol. 185, 165–184. Thomas, B.L., Tucker, A.S., Qui, M., Ferguson, C.A., Hardcastle, Z., Rubenstein, J.L., Sharpe, P.T., 1997. Role of Dlx-1 and Dlx-2
genes in patterning of the murine dentition. Development 124, 4811–4818. Willott, J.F., 1986. Effects of aging, hearing loss, and anatomical location on thresholds of inferior colliculus neurons in C57BL/6 and CBA mice. J. Neurophysiol. 56, 391–408.
Hearing Research
Research paper
Severe hearing loss in Dlx1 mutant mice Daniel B. Polley
a,1
, Inma Cobos b, Michael M. Merzenich a, John L.R. Rubenstein
b,*
a
b
Coleman Memorial Laboratory, W.M. Keck Foundation Center for Integrative Neuroscience, Department of Otolaryngology, University of California, San Francisco, CA 94143, USA Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, CA 94158, USA Received 15 July 2005; received in revised form 16 February 2006; accepted 16 February 2006
Abstract The Dlx homeobox gene family participates in regulating middle and inner ear development. A significant role for Dlx1, in particular, has been demonstrated in the development of the middle ear ossicles, but the functional consequences of Dlx1 gene mutation on hearing thresholds has not been assessed. The present study characterizes auditory brainstem responses to click and tonal stimuli in a non-lethal variant of a Dlx1 gene knockout. We found that peripheral hearing thresholds for click and tonal stimuli were significantly elevated in homozygous Dlx1 knockout (Dlx1 / ) compared to both heterozygous (Dlx1+/ ) and wild type (Dlx1+/+) mice. Thus, abnormal morphogenesis of the incus and stapes that has been documented previously with histological measures is now known to result in a severe peripheral hearing deficit. Ó 2006 Elsevier B.V. All rights reserved. Keywords: ABR; Branchial arch; Incus; Stapes; Mouse; Ossicles; Development
1. Introduction Dlx1, 2, 3, 4, 5 & 6 genes encode homeodomain transcription factors that have essential functions in multiple regions of prenatal and postnatal mammals. The mouse Dlx genes are expressed in multiple tissues including the embryonic brain, face and limbs (Depew et al., 1999; Depew et al., 2002; Ellies et al., 1997; Ferrari and Kosher, 2002; Panganiban and Rubenstein, 2002; Qiu et al., 1997). In addition to regulating early limb morphogenesis, Dlx genes regulate patterning and differentiation of skeletal tissues (Acampora et al., 1999; Beverdam et al., 2002; Depew et al., 1999; Depew et al., 2002; Ferrari and Kosher, 2002; Abbreviations: ABR, auditory brainstem response; PCR, polymerase chain reaction; dB SPL, decibels sound pressure level; kHz, kilohertz * Corresponding author. E-mail addresses: [email protected] (D.B. Polley), john. [email protected] (J.L.R. Rubenstein). 1 Present address: Department of Hearing and Speech Sciences, Vanderbilt University, 465 21st Ave. South, 7110 MRB III, Nashville, TN 37232-8548, USA. Tel.: +615 343 0577; fax: +615 936 3745. 0378-5955/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.02.008
Harris et al., 2003; Hassan et al., 2004; Lezot et al., 2002; Thomas et al., 1997). Within the otic vesicle, different Dlx genes are expressed in distinct patterns that correlate with its functional subdivisions. Dlx5 expression is detected in the lagena macula and the cochlear and vestibular nerves. Dlx1 and Dlx2 expression, in contrast, have not been detected in the otic vesicle; but are found in non-neuronal cells of the cochleovestibular ganglion and nerves (Brown et al., 2005). Although Dlx1 has been known to regulate development of the middle ear for some time (Qiu et al., 1997), the specific consequences of Dlx1 mutations on middle ear morphogenesis has only been described recently (Depew et al., 2005). The more detailed analysis of middle ear morphology in Dlx1 / mice performed in this recent study revealed that the caudal processus brevis and processus longus of the incus can develop as unattached to the rest of the incus. In approximately 50% of the cases, the stapes is hypoplastic and lacks a foramen (through which the stapedial artery normally traverses); in the cases when the foramen forms, it is usually asymmetrically placed. It was
D.B. Polley et al. / Hearing Research 214 (2006) 84–88
also shown that the effects of Dlx1 mutation are titrated according to the number of functional alleles. Mice in the heterozygous state exhibited abnormalities in the basal lamina of the ala temporalis in a similar fashion to the homozygous mice, but the formation of the ossicles appeared normal. The present study seeks to expand our understanding of Dlx1 gene mutations on middle ear development by providing a functional analysis of hearing thresholds to compliment previous histological analyses.
85
acteristic of that seen at higher intensities. In some cases, a response could not be detected at the highest intensity tested. These cases are marked separately in Fig. 2A–E. For the purposes of statistical comparisons, however, these cases were scored as having a response threshold equal to 70 dB SPL. Thresholds were compared for each stimulus type with a Mann–Whitney U statistic. All recordings and analyses were performed blind with respect to genotype.
2. Materials and methods Dlx1 / mice used in this study were initially made using 129-strain ES cells. The mice were initially carried on the C57BL/6J strain, and then out-crossed for at least 5 generations onto the CD1 strain to increase their viability. PCR genotyping followed the methods described in Qiu et al. (1997). Fifteen mice (N = 5 for Dlx1 / , Dlx1+/ , and Dlx1+/+groups) of the C57BL/6J;CD1 hybrid strain were used in this study. All mice were 7–8 weeks at the time of recording. Prior to electrode placement, animals were anesthetized with ketamine (100 mg/ kg i.p.) and medetomidine (0.3 mg/kg i.p.). Body temperature was maintained near 37.5 °C with a rectal probe and homeothermic blanket (Harvard Instruments). Auditory brainstem response (ABR) measurements were performed in a sound-attenuated chamber (Acoustic Systems). ABRs were recorded with silver wire electrodes (0.13 mm diameter, A–M systems) threaded through the skin at three locations: The positive electrode was placed directly behind the right ear over the bulla, the negative electrode over the vertex, and the ground electrode was placed over the neck muscles. ABR signals were acquired, filtered, amplified, and analyzed with equipment and software manufactured by Tucker-Davis Technologies (Alachua, FL). Click and tone pip stimuli were presented with a freefield speaker (Vifa) positioned 22 cm from the external auditory opening. Tone pips (1.2 ms duration, 0.2 ms raised cosine ramps) were presented at 4, 8, 16 and 32 kHz. Acoustic calibration was performed with a microphone (Bruel & Kjaer) to ensure that tone and click stimuli were presented at the specified intensity. Auditory thresholds were obtained for click and tonal stimuli. Click thresholds were determined by presenting 500 click stimuli (10/s) at 50 dB SPL and reducing the sound level in 5 dB SPL steps until the response pattern was no longer visible. If no response was visible at 50 dB SPL, the sound level was increased in 5 dB SPL steps up to a maximum of 70 dB SPL. Tone thresholds were determined in a similar fashion with the exception that sound levels were changed in 10 dB SPL steps rather than 5 dB SPL steps and ABR traces were generated with 1000 stimulus presentations rather than 500. The entire range of sound levels was presented for one animal in each group to obtain a complete set of ABR records for illustrative purposes (Fig. 1). Auditory thresholds were defined for each stimulus as the lowest sound intensity capable of eliciting a response pattern char-
Fig. 1. Determination of ABR thresholds. Representative ABR patterns in a Dlx+/+ mouse (A), Dlx+/ mouse (B), and Dlx / mouse (C) elicited by a 8 kHz tone stimulus presented at sound levels ranging from 0–70 dB SPL. All traces are shown on the same fixed amplitude scale indicated by the scale bar in (A). Open arrows indicate the time at which the sound stimulus reaches the external auditory opening. Closed arrows indicate the threshold response for each mouse. Inset: the threshold response is presented on a normalized scale to illustrate the preservation of the response pattern despite the reduction in absolute amplitude.
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Fig. 2. Quantification of ABR thresholds. (A–E) Response thresholds measured with click and tonal stimuli. Each dot represents the threshold measurement obtained from a single mouse. (F) Mean ± standard error ABR thresholds for Dlx+/+mice (open circles), Dlx+/ mice (gray triangles), and Dlx / mice (black squares) for each stimulus type. Asterisks indicate significant differences (p < 0.025) between Dlx+/+and Dlx / groups based on the Mann–Whitney U statistic.
Confidence intervals were set according to the Bonferroni adjustment for multiple comparisons. 3. Results The ABR waveforms evoked by an 8 kHz tone pip are shown for one representative mouse from each group in Fig. 1. Auditory stimulus presentation evoked the characteristic multi-peaked waveform that became progressively weaker as sound level decreased. While the overall shape of the ABR is similar between groups, the 8 kHz response threshold was substantially elevated in Dlx1 / mice (Fig. 1C) compared to Dlx1+/ (Fig. 1B) and Dlx1+/+ (Fig. 1A) mice. We also observed elevated response thresholds in Dlx1 / mice for other types of auditory stimuli. Response thresholds for each stimulus type are shown in Fig. 2A–E for each individual mouse. Click-evoked response thresholds were 46 dB SPL higher in Dlx1 / mice than
Dlx1+/+ controls (mean ± standard error = 58.0 ± 2.6 dB SPL vs. 12.0 ± 2.0 dB; p < 0.01; Fig. 2A) yet click thresholds in Dlx1+/ mice were not significantly different than Dlx1+/+ controls (15.0 ± 2.04; p = 0.3). Note that a clickevoked threshold could not be calculated for one Dlx1+/ mouse because it died prematurely. Stimulus evokedresponses to 4 kHz tones were not observed at any sound level in any Dlx1 / mice. Accordingly, we observed a significant difference in 4 kHz response threshold between Dlx1+/+ and Dlx1 / mice (Dlx1+/+ = 40.0 ± 3.16 dB SPL; p < 0.005; Fig. 2B) but not between Dlx1+/+ and Dlx1+/ mice (Dlx1+/ mice = 44.0 ± 2.45 dB SPL; p = 0.34). Response thresholds for 8 kHz tones were 48 dB SPL higher on average in Dlx1 / mice compared to Dlx1+/+ controls (64.0 dB SPL ± 2.45 vs. 16.0 ± 2.45 dB; p < 0.01; Fig. 2C) but were not significantly different between Dlx1+/+ and Dlx1+/ mice (22.0 ± 3.74 dB; p = 0.21). Similarly, 16 kHz response thresholds in Dlx1 / mice were 32 dB SPL higher on
D.B. Polley et al. / Hearing Research 214 (2006) 84–88
average than Dlx1+/+ controls (48.0 ± 3.74 dB SPL vs. 16.0 ± 2.45 dB; p < 0.01; Fig. 2D) but were not different than Dlx1+/ mice (22.0 ± 3.74 dB; p = 0.21). Response thresholds at 32 kHz, in contrast, were not significantly different between Dlx1+/+mice (66.0 ± 4.0 dB) and Dlx1 / mice (66.0 ± 2.5 dB; p = 0.7) or Dlx1+/ mice (56.0 ± 6.0 dB; p = 0.19; Fig. 2E). 4. Discussion In summary, we have shown that Dlx / mice exhibit significantly elevated hearing thresholds for frequencies less than 32 kHz compared to Dlx1+/+ controls. The lack of a threshold difference at 32 kHz may be attributable to a diminished sensitivity to high-frequency sounds that is known to occur in C57BL/6J mice irrespective of the Dlx1 mutation rather than a partial sparing of hearing thresholds in Dlx / mice (Henry and Chole, 1980; Willott, 1986). Although the onset of age-related hearing loss in C57B6 mice does not typically occur until mice are several months older than those used in this study, we used C57B6/CD1 hybrid mice in which the exact progression of age-related high-frequency hearing loss is unknown. Hearing thresholds in Dlx1+/ mice, in contrast, were statistically indistinguishable from Dlx1+/+ controls although there was a trend for a slightly elevated thresholds for frequencies less than 32 kHz. Abnormal middle ear morphogenesis previously documented in homozygous Dlx1 mutants with histological methods has been shown in the present study to result in severe hearing loss. This hearing loss is likely to be exclusively attributable to middle ear dysfunction as there is no evidence for Dlx1 expression in the inner ear, nor any evidence for abnormal cochlear development in Dlx1 mutants (Depew et al., 2005; Qiu et al., 1997). In addition to their established role in craniofacial development, Dlx genes are also known to regulate various aspects of interneuron migration and survival in the forebrain (Panganiban and Rubenstein, 2002). Specifically, Dlx1 / mice have recently been shown to exhibit a selective loss of somatostatin+ and calretinin+ interneurons in the cerebral cortex and an associated increase in the incidence of generalized electrographic seizures (Cobos et al., 2005). The present study was motivated by an interest in characterizing abnormalities in physiological organization and stimulus processing in the auditory cortex of Dlx1 / mice. Clearly, we could not pursue these experiments given the severe peripheral hearing deficit observed in homozygous mice. Nevertheless, preliminary evidence suggests that the Dlx1+/ mice may also exhibit a cortical phenotype (JLRR, unpublished data). Given that these mice have relatively normal hearing thresholds, Dlx1+/ mice might prove to be a valuable model for future studies interesting in the link between Dlx1 gene expression and the physiological organization of central auditory nuclei.
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