Abrupt Change in Electrophysiological Properties Begins From Postnatal Day 7 Before Hearing Onset in the Developing Mice Auditory Cortical Layer II/III Neurons

OBJECTIVES: In the developing auditory cortex, maturation of electrophysiological properties and cell types before and after hearing onset has been reported previously. However, the exact timing of firing pattern change has not been reported. In this study, firing pattern change was investigated from postnatal day 3 (P3) to P12 in auditory cortical layer II/III neurons to investigate whether firing pattern changes dramatically after a specific point during development. METHODS: ICR mice pups aged from P3 to P12 were sacrificed to obtain 300-mm-thick brain slices containing the primary auditory cortex. From cortical layer II/III neurons, the patterns of action potential firing generated by current injection were examined using whole cell current clamp technique and the characteristics of Na⁺ currents involved in action potential firing were investigated using whole cell voltage clamp technique. RESULTS: From P3 to P6, most cells did not show action potential firing (29 of 46 cells), and some cells responding to current injection showed a single action potential at the initial depolarizing current step (17 of 46 cells). This firing pattern changes from P7. From P7 to P9, cells begin to show regular spiking to current injection. The spiking frequency increased after P10. In studying Na⁺ current with whole cell voltage clamp, Na⁺ current densities increased gradually (32.0±2.0 pA/pF [P3–P6, n=7], 51.2±2.0 pA/pF [P7–P9, n=13], and 69.5±3.7 pA/pF [P10–P12, n=13]) in low external [Na⁺] condition. Na⁺ current recovery was accelerated and inactivation curves shifted to hyperpolarization with age. CONCLUSION: As regular spiking cells were observed from P7 but never from P3 to P6, P7 might be regarded as an important milestone in the development of auditory cortical layer II/III neurons. This change might mainly result from the increase in Na⁺ current density.

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.

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 of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Kanamycin-treated Rats

OBJECTIVES: From our previous study about the weak expressions of potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) in circling mice, we hypothesized that partially damaged cochlea of circling mice might be a cause of the weak expressions of KCC2 or NKCC1. To test this possibility, we reproduced the altered expressions of KCC2 and NKCC1 in the LSO of rats, whose cochleae were partially destroyed with kanamycin. METHODS: Rat pups were treated with kanamycin from postnatal (P)3 to P8 (700 mg/kg, subcutaneous injection, twice a day) and sacrificed for immunohistochemical analysis, scanning electron microscope (SEM) and auditory brain stem response. RESULTS: The SEM study revealed partially missing hair cells in P9 rats treated with kanamycin, and the hearing threshold was elevated to 63.8+/-2.5 dB SPL (4 ears) at P16. Both KCC2 and NKCC1 immunoreactivities were more prominent in control rats on P16. On 9 paired slices, the mean densities of NKCC1 immunoreactivities were 118.0+/-1.0 (control) and 112.2+/-1.2 (kanamycin treated), whereas those of KCC2 were 115.7+/-1.5 (control) and 112.0+/-0.8 (kanamycin treated). CONCLUSION: We concluded that weak expressions of KCC2 and NKCC1 in circling mice were due to partial destruction of cochleae.

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.

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 Ba2+ (30micrometer) or Cs+ (3micrometer). 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.

Tetanus-induced LTD of Developing MNTB-LSO Synapses in Rat is Dependent on Postsynaptic Ca2+

Because synaptic refinement of medial nucleus of trapezoid body (MNTB) - lateral superior olive (LSO) synapses is most active during the first postnatal week and the long term depression (LTD) has been suggested as one of its mechanisms, LTD of MNTB-LSO synapses was investigated in neonatal rat brain stem slices with the whole cell voltage clamp technique. In Mg2+ free condition, tetanus (10 stimuli at 10 Hz for 2 min) in the current clamp mode induced a robust LTD of isolated D, L-APV-sensitive postsynaptic currents (PSCs) for more than 30 min (n=6, 2.4+/-0.4% of the control), while isolated CNQX-sensitive PSCs were not suppressed (n=6, 95.3+/-1.6%). Tetanus also elicited similar LTD in the isolated GABAergic/glycinergic PSCs (n=5, 3.6+/-0.5%) and mixed PSCs (GABAergic/glycinergic/glutamatergic) (n=4, 2.2+/-0.7%). However, such a strong LTD was not observed in the mixed PSCs when 10 mM EGTA was added in the internal solution (n=10), indicating that postsynaptic Ca2+ rise is needed for the strong LTD. This robust LTD might contribute to the active synaptic refinement occurring during the first postnatal week.

Inhibitory Effects of Potassium Channel Blockers on Carbachol-induced Contraction in Rat Detrusor Muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.

The Relationship Between Time Useful Consciousness and Various Physiological Factors in ROKAF Pilots

Hypoxia is a serious aviation problem and will be a source of dangerous aerospace accidents. Hypoxia tolerance has been evaluated by the time of useful consciousness (TUC), subjective symptoms, hypoxic ventilatory responses (HVR), and cardiovascular changes. It is not clear which physiological factors relate to the former parameters and only few studies are occurred in Korea. From 15 May 1998 through 17 September 1999, 929 ROKAF pilots are checked CBC, blood chemistry, health-related physical fitness tests, pulmonary function tests, life-style, and we analyzed the variables showing significant correlation with TUC. Age, total flying time, grade, and smoking duration had negative correlation with TUC. BMI, FVC, TV, and flexibility had positive correlation. But the reasons why fighter pilots showed significantly shorter TUC than transporter pilots are needed more following study. Also further studies must be made on the intervention study for hypoxia tolerance promotion of ROKAF pilots

Norm-referenced standard of health-related physical fitness of ROKAF pilots

BACKGROUND: The present study is undertaken to acquire normative data of health-related physical fitness(HRPF) of Republic of Korea Air Force(ROKAF) aircrews and to provide norm-referenced standard of HRPF. METHODS: From 15 May 1998 through 14 December 1999, 1284 KAF pilots are checked muscular strength, muscular endurance, flexibility, and body fat percentage. RESULTS: The grip strength was relatively maintained, but flexibility, back strength, and muscular endurance were declined according to promotion and aging. And the incidence of obesity determined by bioelectrical impedence analysis was higher than that assessed on the basis of height and weight. CONCLUSION: It is suggested that many pilots who were normal in BMI have abdominal or visceral obesity. Further studies must be made on the health promotion program for the pilots, on the changes in physical fitness, and on the criterion-referenced standard of HRPF of the pilots.

A Study on the Vestibular Function in the Pilots

To investigate the short-term vestibular responses in the pilots, the 4 successive velocity step tests were done on the 25 pilots, who are in active service in ROKAF (Republic of Korea Air Force), and the obtained results were compared with those of the non-pilots. As the repeated rotations might alter the vestibulo-ocular reflex (VOR), sinusoidal harmonic acceleration (SHA) test at a frequency of 0.16 Hz were also given before and after the 4 successive velocity step tests to investigate the changes of gain and phase of VOR. In the 25 pilots (mean age 31 year), the mean values of the slow cumulative eye position (SCEP), the time integral of eye velocity during nystagmus and time constants were gradually reduced with 4 successive trials. These values were significantly greater than those obtained in the 25 non-pilots. The gain of vestibulo-ocular reflex (VOR) induced by SHA test at a frequency of 0.16 Hz decreased from 0.64+/-0.04 to 0.58+/-0.03 in the pilots, while the VOR gain increased from 0.59+/-0.03 to 0.78+/-0.06 in the non-pilots. However, this result did not indicate that the pilots' gains of VOR were out of normal range. In another SHA tests in which the 30 pilots participated, the gains, phases and the symmetries of VOR lied in the very normal range in every test frequencies (0.01, 0.02, 0.04, 0.08, 0.16, 0.32 Hz). From the above results, it might be suggested that the vestibular responses of the pilots are different with those of the non-pilots.

Evidence for adenosine triphosphate (ATP) as an excitatory neurotransmitter in guinea-pig gastric antrum

We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (>20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2x-purinoceptor with alpha, beta-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggestingthe existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP > ATP gtoreq UTP for contraction and alpha, beta-MeATP gtoreq beta, gamma-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas alpha, beta-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.