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 (IK,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 IK,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 IK,n reported in other tissues. Our results show that the delayed rectifier type K+ outward currents, which are not similar to IK,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 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 Ca(2+) 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 Ca(2+) 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.

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.

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.

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) approximately P11 homozygous (cir/cir) circling mice, perfusion with 10 microM DL-APV and 10 microM 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 approximately 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 approximately 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.

Neural induction with neurogenin1 increases the therapeutic effects of mesenchymal stem cells in the ischemic brain.

Mesenchymal stem cells (MSCs) have been shown to ameliorate a variety of neurological dysfunctions. This effect is believed to be mediated by their paracrine functions, since these cells rarely differentiate into neuronal cells. It is of clinical interest whether neural induction of MSCs is beneficial for the replacement therapy of neurological diseases. Here we report that expression of Neurogenin1 (Ngn1), a proneural gene that directs neuronal differentiation of progenitor cells during development, is sufficient to convert the mesodermal cell fate of MSCs into a neuronal one. Ngn1-expressing MSCs expressed neuron-specific proteins, including NeuroD and voltage-gated Ca2+ and Na+ channels that were absent in parental MSCs. Most importantly, transplantation of Ngn1-expressing MSCs in the animal stroke model dramatically improved motor functions compared with the parental MSCs. MSCs with Ngn1 populated the ischemic brain, where they expressed mature neuronal markers, including microtubule associated protein 2, neurofilament 200, and vesicular glutamate transporter 2, and functionally connected to host neurons. MSCs with and without Ngn1 were indistinguishable in reducing the numbers of Iba1+, ED1+ inflammatory cells, and terminal deoxynucleotidyl transferase dUTP nick-end labeling(+) apoptotic cells and in increasing the numbers of proliferating Ki67+ cells. The data indicate that in addition to the intrinsic paracrine functions of MSCs, motor dysfunctions were remarkably improved by MSCs able to transdifferentiate into neuronal cells. Thus, neural induction of MSCs is advantageous for the treatment of neurological dysfunctions.

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.

Early exposure to radiofrequency electromagnetic fields at 1850 MHz affects auditory circuits in early postnatal mice.

In the present study, we measured the spontaneous post synaptic currents (sPSCs) at the post synaptic principle cells of the medial nucleus of the trapezoid body (MNTB) in early postnatal mice after exposure to 1850 MHz radiofrequency electromagnetic fields (RF-EMF). sPSC frequencies and amplitudes were significantly increased in the RF-EMF exposed group. Moreover, the number of synaptic vesicles in the calyx of Held was significantly increased in presynaptic nerve terminals. Following RF-EMF exposure, the number of docking synaptic vesicles in the active zone increased, thereby expanding the total length of the presynaptic active zone in the calyx of Held. These data suggest that the increased sPSCs are a result of greater synaptic vesicle release from presynaptic nerves. However, we found no morphological changes in the inner hair cell ribbon synapses. Further, there were no significant changes in the hearing threshold of the auditory brainstem response at postnatal day 15. Our results indicate that exposure to RF-EMF at an early postnatal stage might directly affect brainstem auditory circuits, but it does not seem to alter general sound perception.

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

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.

Li+ enhances GABAergic inputs to granule cells in the rat hippocampal dentate gyrus.

Defects in GABAergic interneurons are thought to be involved in the pathophysiology of bipolar disorder, and Li+ has been used as a primary therapeutic agent in the treatment. We used the patch clamp technique to investigate whether Li+ affects on spontaneous GABAergic synaptic inputs to granule cells (GCs) in hippocampal dentate gyrus. Extracellularly applied Li+ (25 mM) markedly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs), an effect completely blocked by picrotoxin or bicuculline. Li+ increased sIPSCs frequency in the presence of tetrodotoxin (TTX), but to a lesser extent than its absence. Li+ caused no change in the cumulative amplitude distribution of miniature IPSCs, indicating that a presynaptic mechanism is involved. When TTX was added in the presence of Li+, large-amplitude sIPSCs (>30 pA) were abolished specifically with no effect on small-amplitude sIPSCs (<20 pA). Intracellular Li+ (6 mM) applied via the patch pipette depolarized the resting membrane potential in fast-spiking interneurons, resulting in an increase in spontaneous action potential (AP) firing. This change, however, was not observed in GCs. These results suggest that Li(+)-induced spontaneous AP firing in GABAergic interneurons contributes to the increase in GABAergic synaptic inputs to GCs.

Short-term vestibular responses to repeated rotations in pilots.

INTRODUCTION: [corrected] It has been suggested that vestibular function in pilots differs from that of non-pilots. METHODS: Short-term vestibular responses to repeated rotations were explored in pilots and non-pilots using a rotating chair. Vestibulo-ocular reflex tests were done in 30 pilots (VOR-P) and 30 non-pilots (VOR-NP) at rotation frequencies of 0.01, 0.02,0.04, 0.08, 0.16, and 0.32 Hz. In a separate experiment, VOR tests were done at 0.16 Hz before and after four successive velocity-step tests in twenty-five pilots (STEP-P) and their results were compared with the results of twenty-five non-pilots (STEP-NP) who were studied earlier. RESULTS: VOR-P exhibited normal VOR gains (mean values 0.47, 0.57, 0.63, 0.69, 0.67, and 0.74, respectively, for the frequencies listed above) compared with those of VOR-NP (0.48, 0.54, 0.59, 0.67, 0.72, and 0.79). VOR gain at 0.16 Hz showed little change in STEP-P (0.64 +/- 0.04 to 0.58 +/- 0.032), while it increased in STEP-NP (0.59 +/- 0.03 to 0.78 +/- 0.06). DISCUSSION: VOR gain may be a useful measurement for differentiating pilots' vestibular function from that of non-pilots. After-rotary VOR may be suppressed in pilots.

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.

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 (E(gly)) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted E(gly) 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.

Sustained Fos expression is observed in the developing brainstem auditory circuits of kanamycin-treated rats.

It has been demonstrated that kanamycin treatment during early developmental period induces partial cochlear destruction and enhanced glutamatergic transmission at the medial nucleus of the trapezoid body (MNTB) - the lateral superior olive (LSO) synapses in the superior olivary complex (SOC). As c-fos was expected to be expressed in the SOC by kanamycin-induced cochlear damage, the expression of c-fos protein (Fos) was investigated using immunohistochemistry in kanamycin-treated rat pups. In the control rat pups less than postnatal (P) day 9 in age, Fos-like immunoreactivity (Fos-IR) was transiently observed in the MNTB and LSO on P6, but disappeared on P9, which reflects a physiologic process. In contrast, in kanamycin-treated rats, Fos-IR was consistently observed through P9. Because a significant increase in terminal uridine deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) and glial fibrillary acidic protein (GFAP) IR was not demonstrated in the MNTB and LSO of kanamycin-treated rats, the increased Fos-IR does not appear to indicate an ongoing pathologic process, but may be related to the increased activity caused by the disturbance in excitatory and inhibitory balance between brainstem auditory circuits.

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.

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 (<2 mM) of TEA and 4-AP inhibited phasic and tonic contractions induced by 5 micro M carbachol, whilst the relatively higher concentrations of TEA and 4-AP (>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 microM to 5 microM. 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 microM thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potassium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.