Seung Cheol Ahn

Glutamatergic Transmission Is Sustained at a Later Period of Development of Medial Nucleus of the Trapezoid Body-Lateral Superior Olive Synapses in Circling Mice

Synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) was investigated in circling mice, an animal model for inherited deafness, using the voltage-clamp technique. In postnatal day 9 (P9)∼P11 homozygous (cir/cir) circling mice, perfusion with 10 μm dl-APV and 10 μm CNQX reduced the 10 min average of postsynaptic currents (PSCs) to 8.8 ± 3.0% compared with controls (n = 6). In heterozygous (+/cir) mice in the same age range, the 10 min PSCs average was reduced to 87.5 ± 3.7% compared with controls (n = 7). In P0∼P2 homozygous (cir/cir) and heterozygous (+/cir) mice, the 10 min PSCs averages were 11.0 ± 2.6% (n = 9) and 84.1 ± 4.6% (n = 11), respectively. The effects of a glutamate antagonist mixture were almost the same in single fiber stimulation of P9∼P11 mice, reducing mean PSCs to 5.2 ± 3.1% (homozygous (cir/cir) mice, n = 8) and 78.3 ± 4.3% (heterozygous (+/cir) mice, n = 12). Immunohistochemical study revealed that glycine receptor (GlyR) immunoreactivity in heterozygous (+/cir) mice was more prominent than in homozygous (cir/cir) mice, while immunoreactivities of NR1 and NR2A-type NMDAR of P16 homozygous (cir/cir) mice were more prominent than in heterozygous (+/cir) mice of the same age. No significant difference was found in the immunoreactivity of NR2B-type NMDAR. These results indicate that glutamatergic transmission is sustained at a later period of developing MNTB–LSO synapses in homozygous (cir/cir) mice.

Glutamate co-transmission from developing medial nucleus of the trapezoid body - Lateral superior olive synapses is cochlear dependent in kanamycin-treated rats

Cochlear dependency of glutamate co-transmission at the medial nucleus of the trapezoid body (MNTB)--the lateral superior olive (LSO) synapses was investigated using developing rats treated with high dose kanamycin. Rats were treated with kanamycin from postnatal day (P) 3 to P8. A scanning electron microscopic study on P9 demonstrated partial cochlear hair cell damage. A whole cell voltage clamp experiment demonstrated the increased glutamatergic portion of postsynaptic currents (PSCs) elicited by MNTB stimulation in P9-P11 kanamycin-treated rats. The enhanced VGLUT3 immunoreactivities (IRs) in kanamycin-treated rats and asymmetric VGLUT3 IRs in the LSO of unilaterally cochlear ablated rats supported the electrophysiologic data. Taken together, it is concluded that glutamate co-transmission is cochlear-dependent and enhanced glutamate co-transmission in kanamycin-treated rats is induced by partial cochlear damage.

Immunohistochemical localization of calbindin D28-k, parvalbumin, and calretinin in the cerebellar cortex of the circling mouse

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.

Characterization of Ionic Currents in Human Neural Stem Cells

The profile of membrane currents was investigated in differentiated neuronal cells derived from human neural stem cells (hNSCs) that were obtained from aborted fetal cortex. Whole-cell voltage clamp recording revealed at least 4 different currents: a tetrodotoxin (TTX)-sensitive Na(+) current, a hyperpolarization-activated inward current, and A-type and delayed rectifier-type K(+) outward currents. Both types of K(+) outward currents were blocked by either 5 mM tetraethylammonium (TEA) or 5 mM 4-aminopyridine (4-AP). The hyperpolarization-activated current resembled the classical K(+) inward current in that it exhibited a voltage-dependent block in the presence of external Ba(2+) (30microM) or Cs(+) (3microM). However, the reversal potentials did not match well with the predicted K(+) equilibrium potentials, suggesting that it was not a classical K(+) inward rectifier current. The other Na(+) inward current resembled the classical Na(+) current observed in pharmacological studies. The expression of these channels may contribute to generation and repolarization of action potential and might be regarded as functional markers for hNSCs-derived neurons.

Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice.

Vesicular glutamate transporter 3 (VGLUT3) plays a major role in hearing, and mice lacking the VGLUT3 are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapses. However, whether VGLUT3 is expressed normally in the cochleae of developing circling mice (homozygous (cir/cir) mice), the animal model for human deafness type DFNB6, has not been established. In this study, we investigated the developmental expression of VGLUT3 in cochlear inner hair cells (IHCs) and spiral ganglion cells (SGCs) of homozygous (cir/cir) mice from postnatal day (P)1 to P14 using immunofluorescence (IF) staining and Western blot. VGLUT3 immunoreactivity (IR) and protein expression increased progressively with age in homozygous (cir/cir) and control mice (heterozygous (+/cir) mice and ICR mice). The rank order of VGLUT3 IR in IHCs and SGCs in P14 mice was homozygous (cir/cir) mice = heterozygous (+/cir) mice > ICR mice. The rank order of total protein expression was homozygous (cir/cir) mice > heterozygous (+/cir) mice = ICR mice at P14. IF staining and Western blot analysis indicated that developmental VGLUT3 expression in cochleae was most prominent in homozygous (cir/cir) mice. The possible contribution of VGLUT3 upregulation in the cochlear degeneration is discussed.

Reinvestigation of cochlear pathology in circling mice.

The main causes of early hearing deficit in circling mice have been reported to be early degeneration of the organ of Corti and deterioration of spiral ganglion neurons. As an exact cochlear pathology is essential to explain our previous results regarding the auditory brainstem circuits of developing circling mice, we reinvestigated the cochlear pathology in developing circling mice (14, 22, and 38 days old). It has been reported that the organ of Corti in circling mice completely degenerates as early as postnatal day (P) 21 and that circling mice are deaf by P18. Although we confirmed that circling mice were deaf at P15 and that hair bundles of outer hair cells were defective at P18, complete degeneration of the organ of Corti was not observed by P38 in circling mice. At P22, the type I cell-like spiral ganglion cell density in circling mice was reduced to 78% of that of control mice (ICR mice), but it was not significantly different from that of other control mice (heterozygous (+/cir) mice, littermates of circling mice) that could hear at P22. Our data suggest that other factors, such as absence of neurotransmitter release from inner hair cells, should be considered to explain the early hearing deficit observed at P15 in circling 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 µM 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.

Glycine-induced currents are insensitive to the glycine receptor α1 subunit-specific blocker, cyanotriphenylborate, in older circling mice.

The pharmacologic characteristics of glycine receptors (GlyRs) in the lateral superior olive (LSO) of circling mice, animal model for inherited deafness, were investigated using a GlyR α1 subunit-specific receptor blocker (cyanotriphenylborate [CTB]). There was a statistically significant age-dependent increase in the antagonistic effect of CTB in heterozygous (+/cir) mice. In postnatal (P)0-P3 heterozygous (+/cir) mice, glycine currents evoked by glycine puffs were reduced to 20.4±2.6, 37.1±3.1, and 63.9±2.5% at 0.1, 1, and 10 μM CTB (n=13) compared to controls, while the glycine currents were reduced to 22.3±3.5, 52.9±4.1, and 78.3±3.5% at 0.1, 1, and 10 μM CTB (n=7) in P8-P12 heterozygous (+/cir) mice. In contrast, the antagonistic effect of CTB was not strong and even less than that of younger animals in older homozygous (cir/cir) mice. In P0-P3 homozygous (cir/cir) mice, the extent of inhibition was 20.2±3.7, 37.8±4.3, and 66.8±4.2% at 0.1, 1, and 10 μM CTB (n=6) compared to controls, while the extent of inhibition was 18.7±2.4, 28.1±3.9, and 39.1±8.2% (n=6) in P8-P12 homozygous (cir/cir) mice. The age-dependent decrease in the antagonistic effect of CTB indicates the abnormal development of the α1 subunit-containing GlyRs in homozygous (cir/cir) mice.

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 Vhalf 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.

Long term depression of MNTB-LSO synapses is expressed postsynaptically in developing circling mice.

Early onset long term depression (LTD) during the first postnatal week has rarely been demonstrated at the medial nucleus of trapezoid body (MNTB) - lateral superior olive (LSO) synapses in spite of many favorable conditions, such as depolarizing synapses and glutamate co-release from MNTB terminals. Thus, we tested the early expression of LTD at MNTB-LSO synapses during the first postnatal week using circling mice, whose main transmitter is glutamate at MNTB-LSO synapses. Tetanic stimulation on MNTB elicited LTD of postsynaptic currents recorded at LSO neurons in P0-P3 homozygous (cir/cir) mice (45.8 ± 0.3% of the control, n = 7) and heterozygous (+/cir) mice (43.3 ± 0.4% of the control, n = 7). The magnitude of LTD decreased in P8-P12 heterozygous (+/cir) mice (84.5 ± 0.3% of the control, n = 7), but was maintained in P8-P12 homozygous (cir/cir) mice (38.2 ± 0.3% of the control, n = 9). Glutamatergic LTD observed in homozygous (cir/cir) mice and glycinergic LTD observed heterozygous (+/cir) mice showed similar pattern of change. As currents induced by the pressure application of glycine on LSO neurons were reduced by tetanic stimulation in P0-P3 heterozygous (+/cir) mice, LTD was thought to occur at postsynaptic sites. Our results suggest that LTD might occur in vivo and participate in the synaptic silencing and strengthening of MNTB-LSO synapses, which is most active during the first postnatal week.