Forskolin and protein kinase inhibitors differentially affect hair cell potassium currents and transmitter release at the cytoneural junction in the isolated frog labyrinth.

The post-transductional elaboration of sensory input at the frog semicircular canal has been studied by correlating the effects of drugs that interfere with phosphorylation processes on: (i) potassium conductances in isolated hair cell and (ii) transmitter release at the cytoneural junction in the intact labyrinth. At hair cells, delayed potassium currents (IKD) undergo voltage- and time-dependent inactivation; inactivation removal requires ATP, is sensitive to kinase blockade, but is unaffected by exogenous application of cyclic nucleotides. We report here that forskolin, an activator of endogenous adenylyl cyclase, enhances IKD inactivation removal in isolated hair cells, but produces an overall decrease in IKD amplitude consistent with the direct blocking action of the drug on several families of K channels. In the intact labyrinth, forskolin enhances transmitter release, consistent with such depression of K conductances. Kinase blockers - H-89 and KT5823 - have been shown to reduce IKD inactivation removal and IKD amplitude at isolated hair cells. In the labyrinth, the effects of these drugs on junctional activity are quite variable, with predominant inhibition of transmitter release, rather than the enhancement expected from the impairment of K currents. The overall action of forskolin and kinase inhibitors on K conductances is similar (depression), but they have opposite effects on transmitter release: this indicates that some intermediate steps between the bioelectric control of hair cell membrane potential and transmitter release are affected in opposite ways and therefore are presumably regulated by protein phosphorylation.

Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio).

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

Frequency perception and cell injury in the semicircular canal caused by gentamicin.

OBJECTIVE: The frequency characteristics of the vestibular organ have gained notice in recent years, but the morphologic basis was unknown. This study investigated the gentamicin-induced damage of frequency-selective perception of the horizontal semicircular canal and its morphologic basis. METHODS: Eighty guinea pigs were randomly divided into four groups, one control group and three experimental groups. The experimental animals received gentamicin subcutaneously for 1 to 3 weeks. Short-latency vestibular evoked potentials evoked by 0.5 and 10 Hz step rotation stimuli following drug administration were recorded, and then the crista ampullaris of the horizontal semicircular canals was investigated by scanning and transmission electron microscopy. RESULTS: Damage to hair cells of the crista ampullaris is concentrated at the apex area first and then extends to the peripheral area of the vestibular crista ampullaris when the gentamicin administration time increased. When only the hair cells at the apex area are damaged, the high-frequency (10 Hz) rotation perception of the crista ampullaris of the horizontal semicircular canal was injured, but perceptions to 0.5 Hz step rotation stimulation remained normal. CONCLUSION: Gentamicin mainly affects the high-frequency perception function of the crista ampullaris of the horizontal semicircular canal. The hair cells at the central apex area of the crista ampullaris might be responsible for high-frequency rotation perception function.

Morphological change of the cupula due to an ototoxic agent: a comparison with semicircular canal pathology.

CONCLUSION: The cupula shows various degrees of changes after gentamicin (GM) injection into the inner ear, with or without damage of the sensory cells. This cupula change may be a part of the etiology of peripheral vertigo, and is also potentially one of the mechanisms of reduced caloric response. OBJECTIVES: To observe the morphological changes of the cupula after injecting GM in the frog inner ear and to compare the changes of the cupula with those of the ampullary sensory cells. METHODS: We injected 300 microg (7.5 microl) of GM into the inner ear of 30 bullfrogs (Rana catesbeiana) using a microsyringe under ether anesthesia. The same amount of saline was injected into the other ear as control. The cupulae were observed at 3, 7, and 14 days after GM injection by stereoscopic microscope. The ampullae were fixed, and the sensory cells were assessed using a scanning electron microscope (SEM). The correlation between the changes in the cupula and sensory cells was evaluated using our own scale. RESULTS: In over half of the cupulae in the 7- and 14-day groups, cupula changes such as shrinkage were observed. In about 50% of the total cases, the degree of cupula and sensory cell change correlated in the two groups. In the 14-day group, these changes were more marked. However, there were cases in which the changes of the cupula and sensory cells did not correlate, indicating that the cupula alone can sustain changes without sensory cell damage.

Analysis of pre- and postsynaptic activity in the frog semicircular canal following ototoxic insult: differential recovery of background and evoked afferent activity.

Frogs were treated with a single dose of gentamicin administered intraotically to produce severe degeneration of posterior semicircular canal hair cells and to evaluate the time course of functional damage and recovery both at pre- and postsynaptic level. In isolated canal preparations the endoampullar potential, which reflects the summed receptor potentials of crista hair cells, was progressively reduced in amplitude and completely abolished 6 days after gentamicin treatment. At this time the crista epithelium was devoid of hair cells. The recovery of the endoampullar potential began around 9 days after the ototoxic insult and its amplitude progressively increased to reach, after 20 days, values close to those observed in control experiments. The endoampullar potential amplitude was related to the degree of hair cell regeneration in the crista epithelium. Consistent with the presynaptic damage, the slow generator potential (representing the summed miniature excitatory postsynaptic potential [mEPSP] activity of all posterior nerve fibres) and the resting and evoked spike discharge recorded from the whole ampullar nerve were abolished 6 days after gentamicin treatment. The recovery of the background and evoked afferent activity showed different behaviours. Background spike activity became detectable around 8 days after the ototoxic insult, but was not modulated by canal stimulation at this time, and no generator potential was detected. Moreover, the resting spike frequency fully recovered and reached control values around 15 days after gentamicin treatment, whereas the evoked activity attained normal values only 20 days after the ototoxic insult. These results were confirmed by intracellular recordings from single afferent fibres of the ampullar nerve in intact labyrinth preparations. Absence of any resting and evoked discharge was the most common pattern observed in the early period from 7 to 8 days after gentamicin treatment. Fifty-five percent of impaled afferents were silent while the others showed low resting frequencies of mEPSPs and spikes, and were unresponsive to canal rotation. In the intermediate period from 14 to 15 days after gentamicin treatment, background mEPSP and spike frequencies approached those evaluated in control experiments, but the frequencies of the evoked mEPSPs and spikes were clearly lower than in controls. In the late period, from 18 to 20 days after the ototoxic insult, the impaled afferents showed normal evoked mEPSP and spike frequencies. The present data indicate that the frog semicircular canal completely recovers its pre- and postsynaptic activity following severe ototoxic insult. During the regeneration process, the cytoneural junction regains function and the resting discharge reappears before recovery of mechanoelectrical transduction.