Ernest J. Moore

Mercury (Hg2+) suppression of potassium currents of outer hair cells

The heavy metal mercury (Hg(2+)) is an insidious environmental pollutant that causes toxic effects on sensory systems. It is well known that the group IIB divalent cation Hg(2+) is an inhibitor of the group I monovalent potassium (K(+)) cation pore-forming channel in several biological preparations. Here, we used the whole cell patch clamp technique on freshly isolated outer hair cells (OHCs) of the guinea pig cochlea to record outward K(+) currents and inward K(+) currents treated with mercuric chloride (HgCl(2)). HgCl(2) affected K(+) currents in a voltage- and dose-dependent manner. The effects of HgCl(2) at 1.0-100 microM are more pronounced on onset peak current than on steady-state end current. HgCl(2) depolarized also the resting membrane potential. Although the effect of HgCl(2) at 1.0 microM was partially washed out over several minutes, the effects at 10 and 100 microM were irreversible to washout. Since K(+) channels of OHCs are targets for HgCl(2) ototoxicity, this may lead to auditory transduction problems, including a loss in hearing sensitivity. A better understanding of fundamental mechanisms underlying K(+) channelopathies in OHCs due to HgCl(2) poisoning may lead to better preventive or therapeutic agents.

Sodium and Potassium Currents of Type I Spiral Ganglion Cells from Rat

Ion channel activity of acutely dissociated type I spiral ganglion cells isolated from rats was investigated using the whole-cell variation of the patch clamp technique. Tetrodotoxin-sensitive sodium current and tetraethylammonium-sensitive potassium current were recorded. With a holding potential of -80 mV, peak sodium currents were generated by depolarizations to membrane potentials more positive than -50 mV. Potassium currents were elicited at membrane potentials more positive than -40 mV. Some cells which did not exhibit sodium current exhibited delayed rectifier potassium currents. Steady-state sodium channel inactivation curve yielded a slope of 12 mV and a half-inactivated potential of -83 mV. Recovery from inactivation of the sodium channel proceeded with a fast and slow time course; recovery began as early as 0.8 ms and proceeded with a time constant of 7.5 ms. It is concluded that type I spiral ganglion cells are endowed with sodium and potassium channels which are responsible for generation and propagation of auditory nerve action potentials.

Kcnq1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish.

BackgroundKCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ 1), epilepsy in adults (KCNQ 2/3), benign familial neonatal convulsions in children (KCNQ 3), and hearing loss or tinnitus in humans (KCNQ 4, but not KCNQ 5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues.ResultsOverall expression of the kcnq x channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues.ConclusionsWe conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.

Inhibition of K+ currents of outer hair cells in guinea pig cochlea by fluoxetine

The effects of fluoxetine (Prozac), a widely used antidepressant drug, on K+ channel in outer hair cells isolated from guinea pig cochlea were studied using the whole-cell patch clamp technique. Fluoxetine potently inhibited leak K+ currents with an IC50 of 0.78 microM. The inhibition was reversible and voltage-independent. At 45- to 103-fold higher concentrations than the plasma levels, fluoxetine reversibly blocked voltage-activated K+ currents. Kinetics of the current in the presence of fluoxetine resembled the control current, and the inhibition was not use-dependent. Neither the activation curve nor the reversal potential was affected by fluoxetine. This inhibition was voltage-dependent with an electric distance (delta value) of the binding site of at least 26% of the membrane field from the cytoplasmic side. Use-independent inhibition suggests that fluoxetine blocks the channel before its opening or instantly blocks the open channel. This is the first study of the action of this compound on K+ channel of outer hair cells of the mammalian inner ear. We conclude that the block of the leak K+ currents can occur at therapeutic levels of fluoxetine. Since the voltage-activated K+ currents are not potently blocked by fluoxetine, this action might not be related to its antidepressant action or adverse effects.

An M-Like Potassium Current in the Guinea Pig Cochlea

Potassium M currents play a role in stabilizing the resting membrane potential. These currents have previously been identified in several cell types, including sensory receptors. Given that maintaining membrane excitability is important for mechano-electrical transduction in the inner ear, the presence of M currents was investigated in outer hair cells isolated from the guinea pig hearing organ. Using a pulse protocol designed to emphasize M currents with the whole-cell patch-clamp technique, voltage- and time-dependent, non-inactivating, low-threshold currents (the hallmarks of M currents) were recorded. These currents were significantly reduced by cadmium chloride. Results from RT-PCR analysis indicated that genes encoding M channel subunits KCNQ2 and KCNQ3 are expressed in the guinea pig cochlea. Our data suggest that guinea pig outer hair cells express an M-like potassium current that, following sound stimulation, may play an important role in returning the membrane potential to resting level and thus regulating outer hair cell synaptic mechanisms.

An in vitro model for testing drugs to treat tinnitus

Tinnitus affects approximately 50 million people in the USA alone, with 10 million being highly debilitated. Pharmacotherapy for tinnitus is still in emerging stages due to time consuming clinical trials and/or animal experiments. We tested a new cellular model where induced rapid neuronal firing or spiking was used as a mimic for the type of aberrant activity that may occur in tinnitus. Spontaneously active auditory cortical networks growing on microelectrode arrays were exposed to pentylenetetrazol (PTZ), a proconvulsant and an antagonist of GABA(A) receptor, which is implicated in tinnitus. Auditory cortical networks were then exposed to experimental tinnitus drugs linopirdine (Dup966, a potassium channel blocker), L-carnitine (an antioxidant), or selective Ca(2+) channel antagonists pregabalin (Lyrica), or gabapentin (Neurontin) at various concentrations. PTZ increased spike rate by 139.6±27% and burst rate by 129.7±28% in auditory cortical networks with a phenotypic high firing of excitable neurons. Reductions of increased activity were observed to varying degrees using the experimental tinnitus drugs. The potency of the drugs was linopirdine (EC(50): 176±7.0 μM)>L-carnitine (EC(50): 1569±41 μM)>pregabalin (EC(50): 8360±340 μM), >gabapentin, with 34.2±7.5% efficacy (EC(50): 2092±980 μM). These studies provide proof of principle for the use of auditory cortical networks on microelectrode array as a feasible platform for semi-high throughput application for screening of drugs that might be used for the treatment of tinnitus.

Auditory Cortex Neurons: Primary Culture and Ion Channel Activity in Rat

We have developed a primary dissociated cell culture of the fetal (E17) and post-natal (P0-P10) rat auditory cortex. Pyramidal and non-pyramidal cells had a mean cross-sectional diameter of 12.73 +/- 1.80 microns (mean +/- S.D., n = 25) and 17.58 +/- 1.67 microns (mean +/- S.D., n = 10), respectively, measured at 6 days in culture. These cells were viable for as long as 18-21 days. They expressed voltage-gated sodium and potassium channel currents as early as one day in culture, and at various phases in cell culture. Sodium current, activated at membrane potentials more positive than -60 mV, displayed fast activation and inactivation kinetics. Fifty percent inactivation of sodium channels occurred at a pre-pulse potential of -63 mV. Delayed rectifier potassium channels were activated at potentials positive to -40 mV. Large hyperpolarizing constant current pulses elicited anode break action potentials, and large depolarizing constant current pulses exhibited rectification indicative of the delayed rectifying potassium channel activity.

Safety of Autologous Umbilical Cord Blood Therapy for Acquired Sensorineural Hearing Loss in Children

Background and Objectives Sensorineural hearing loss (SNHL) in children is associated with neurocognitive morbidity. The cause of SNHL is a loss of hair cells in the organ of Corti. There are currently no reparative treatments for SNHL. Numerous studies suggest that cord blood mononuclear cells (human umbilical cord blood, hUCB) allow at least partial restoration of SNHL by enabling repair of a damaged organ of Corti. Our objective is to determine if hUCB is a safe treatment for moderate to severe acquired SNHL in children. Subjects and Methods Eleven children aged 6 months to 6 years with moderate to severe acquired SNHL were treated with intravenous autologous hUCB. The cell dose ranged from 8 to 30 million cells/kg body weight. Safety was assessed by measuring systemic hemodynamics during hUCB infusion. Infusion-related toxicity was evaluated by measuring neurologic, hepatic, renal and pulmonary function before and after infusion. Auditory function, auditory verbal language assessments and MRI with diffusion tensor imaging (DTI) were obtained before and after treatment. Results All patients survived, and there were no adverse events. No infusionrelated changes in hemodynamics occurred. No infusion-related toxicity was recorded. Five subjects experienced a reduction in auditory brainstem response (ABR) thresholds. Four of those 5 subjects also experienced an improvement in cochlear nerve latencies. Comparison of MRI with DTI sequences obtained before and after treatment revealed increased fractional anisotropy in the primary auditory cortex in three of five subjects with reduced ABR thresholds. Statistically significant (p<0.05) reductions in ABR thresholds were identified. Conclusions TIntravenous hUCB is feasible and safe in children with SNHL.

Pharmacological response sensitization in nerve cell networks exposed to the antibiotic gentamicin.

&NA; Gentamicin is an aminoglycoside antibiotic that is used in clinical, organismic, and agricultural applications to combat gram‐negative, aerobic bacteria. The clinical use of gentamicin is widely linked to various toxicities, but there is a void in our knowledge about the neuromodulatory or neurotoxicity effects of gentamicin. This investigation explored the electrophysiologic effects of gentamicin on GABAergic pharmacological profiles in spontaneously active neuronal networks in vitro derived from auditory cortices of E16 mouse embryos and grown on microelectrode arrays. Using the GABAA agonist muscimol as the test substance, responses from networks to dose titrations of muscimol were compared in the presence and absence of 100 &mgr;M gentamicin (the recommended concentration for cell culture conditions). Spike‐rate based EC50 values were generated using sigmoidal fit concentration response curves (CRCs). Exposure to 100 &mgr;M gentamicin exhibited a muscimol EC50±S.E.M. of 80±6 nM (n=10). The EC50 value obtained in the absence of gentamicin was 124±11 nM (n=10). The 35% increase in potency suggests network sensitization to muscimol in the presence of gentamicin. Action potential (AP) waveform analyses of neurons exposed to gentamicin demonstrated a concentration‐dependent decrease in AP amplitudes (extracellular recordings), possibly reflecting gentamicin effects on voltage‐gated ion channels. These in vitro results reveal alteration of pharmacological responses by antibiotics that could have significant influence on the behavior and performance of animals.