Inhibition of K⁺ outward currents by linopirdine in the cochlear outer hair cells of circling mice within the first postnatal week

Inhibition of K⁺ outward currents by linopirdine in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), was investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) and ICR mice of the same age (postnatal day (P) 0 –P6) were used as controls. Voltage steps from –100 mV to 40 mV elicited small inward currents (–100 mV~–70 mV) and slow rising K⁺ outward currents (–60 mV ~40 mV) which activated near –50 mV in all OHCs tested. Linopirdine, a known blocker of K⁺ currents activated at negative potentials (I(K,n)), did cause inhibition at varying degree (severe, moderate, mild) in K⁺ outward currents of heterozygous (+/cir) or homozygous (cir/cir) mice OHCs in the concentration range between 1 and 100 µM, while it was apparent only in one ICR mice OHC out of nine OHCs at 100 µM. Although the half inhibition concentrations in heterozygous (+/cir) or homozygous (cir/cir) mice OHCs were close to those reported in I(K,n), biophysical and pharmacological properties of K⁺ outward currents, such as the activation close to –50 mV, small inward currents evoked by hyperpolarizing steps and TEA sensitivity, were not in line with I(K,n) reported in other tissues. Our results show that the delayed rectifier type K⁺ outward currents, which are not similar to I(K,n) with respect to biophysical and pharmacological properties, are inhibited by linopirdine in the developing (P0~P6) homozygous (cir/cir) or heterozygous (+/cir) mice OHCs.

The distribution of calbindin-D28k, parvalbumin, and calretinin immunoreactivity in the inferior colliculus of circling mouse / 대한해부학회지

The circling mice with tmie gene mutation are known as an animal deafness model, which showed hyperactive circling movement. Recently, the reinvestigation of circling mouse was performed to check the inner ear pathology as a main lesion of early hearing loss. In this trial, the inner ear organs were not so damaged to cause the hearing deficit of circling (cir/cir) mouse at 18 postnatal day (P18) though auditory brainstem response data indicated hearing loss of cir/cir mice at P18. Thus, another mechanism may be correlated with the early hearing loss of cir/cir mice at P18. Hearing loss in the early life can disrupt the ascending and descending information to inferior colliculus (IC) as integration site. There were many reports that hearing loss could result in the changes in Ca²⁺ concentration by either cochlear ablation or genetic defect. However, little was known to be reported about the correlation between the pathology of IC and Ca²⁺ changes in circling mice. Therefore, the present study investigated the distribution of calcium-binding proteins (CaBPs), calbindin-D28k, parvalbumin, and calretinin immunoreactivity (IR) in the IC to compare among wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice by immunohistochemistry. The decreases of CaBPs IR in cir/cir were statistically significant in the neurons as well as neuropil of IC. Thus, this study proposed overall distributional alteration of CaBPs IR in the IC caused by early hearing defect and might be helpful to elucidate the pathology of central auditory disorder related with Ca²⁺ metabolism.

Over-expression of myosin7A in cochlear hair cells of circling mice / 한국실험동물학회지

Circling mouse (C57BL/6J-cir/cir) deleted the transmembrane inner ear (Tmie) gene is an animal model for human non-syndromic recessive deafness, DFNB6. In circling mouse, hair cells in the cochlea have degenerated and hair bundles have become irregularity as time goes on. Tmie protein carries out a function of the mechanoelectrical transduction channel in cochlear hair cells. Myosin7a (MYO7A) protein has key roles in development of the cochlear hair bundles as well as in the function of cochlear hair cells. To find whether Tmie protein interacts with MYO7A proteins in the cochlea postnatal developmental stage, we investigated expression of the MYO7A proteins in the cochlear hair cells of circling mice by western blot analysis and whole mount immunofluorescence at postnatal day 5 (P5). The expression of MYO7A showed statistically significant increase in the cochlea of C57BL/6J-+/cir and C57BL/6J-cir/cir mice than that of C57BL/6J-+/+ mice. The MYO7A intensity of the cochlear hair cells also increased in C57BL/6J-+/cir and C57BL/6J-cir/cir mice compared with those of C57BL/6J-+/+ mice. Taken together, the results indicate that Tmie protein may have an important role with MYO7A protein in the development and maintenance of the stereociliary bundles during postnatal developmental stage of the cochlea.

Characteristics of K+ Outward Currents in the Cochlear Outer Hair Cells of Circling Mice within the First Postnatal Week

K+ outward currents in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), were investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) mice of the same age (postnatal day (P) 0 -P6) were used as controls. Similar slow rising K+ currents were observed in both genotypes, but their biophysical and pharmacological properties were quite different. The values of V(half) for activation were significantly different in the heterozygous (+/cir) and homozygous (cir/cir) mice (-8.1+/-2.2 mV, heterozygous (+/cir) mice (n=7) and -17.2+/-4.2 mV, homozygous (cir/cir) mice (n=5)). The inactivation curve was expressed by a single first order Boltzmann equation in the homozygous (cir/cir) mice, while it was expressed by a sum of two first order Boltzmann equations in the heterozygous (+/cir) mice. The K+ current of homozygous (cir/cir) mice was more sensitive to TEA in the 1 to 10 mM range, while the 4-AP sensitivities were not different between the two genotypes. Removal of external Ca2+ did not affect the K+ currents in either genotype, indicating that the higher sensitivity of K+ current to TEA in the homozygous (cir/cir) mice was not due to an early expression of Ca2+ activated K+ channels. Our results suggest that the K+ outward current of developing homozygous (cir/cir) mice OHCs is different in both biophysical and pharmacological aspects than that of heterozygous (+/cir) mice.

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

OBJECTIVES: We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice. METHODS: Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials. RESULTS: Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 microM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9+/-2.6 mV to -66.0+/-1.5 mV [bumetanide], -66.0+/-1.5 mV to -59.8+/-2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1+/-2.9 mV to -62.7+/-2.6 mV [bumetanide], -62.7+/-2.6 mV to -62.1+/-2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice. CONCLUSION: The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

OBJECTIVES: We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice. METHODS: Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials. RESULTS: Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 microM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9+/-2.6 mV to -66.0+/-1.5 mV [bumetanide], -66.0+/-1.5 mV to -59.8+/-2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1+/-2.9 mV to -62.7+/-2.6 mV [bumetanide], -62.7+/-2.6 mV to -62.1+/-2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice. CONCLUSION: The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.

Investigation of Developmental Changes of Convergence Ratios of MNTB-LSO Synapses in Circling Mice / 대한해부학회지

The developmental changes of convergence ratios of medial nucleus of trapezoid body (MNTB) axons to single lateral superior olive (LSO) neuron were investigated using voltage clamp technique in homologous (cir/cir) circling mice, animal model for the congenital deafness with autosomal recessive inheritance. As the developmental reduction of convergence ratio reported in rats indicates the presence of synaptic refinement, we aimed to find out whether the similar reduction of convergence ratio also occurs in circling mice. Heterologous (+/cir) mice were used as control and mice younger than postnatal (P) day 4 or older than P9 were used. The convergence ratios of MNTB axons to single LSO neuron were 29.16+/-2.7 (n=12, P9) in homologous (cir/cir) mice, while they were 37.89+/-3.8 (n=9, P9) in heterozygous (+/cir) mice. The significant changes were observed only in heterozygous (+/cir) mice, which indicated that synaptic refinement of MNTBLSO synapses occurs in heterozygous (+/cir) mice, not in homozygous (cir/cir) mice. Considering homologous (cir/cir) mice being animal model for the congenital deafness, our data might contribute to the understanding of developmental changes of brain stem auditory circuits of congenitally deaf patients.