Recovery of pre- and postsynaptic activity in frog semicircular canal after gentamicin treatment

Inner ear hair cells of lower vertebrates regenerate following physical or chemical insults. In the present study we aimed to define the damage and recovery of the pre and postsynaptic activity in the frog crista ampullaris after ototoxic insult with the antibiotic, gentamicin. Frogs were treated with a single dose of gentamicin sulphate (GM-5mM) administered intraotically. The presynaptic activity was monitored by recording in isolated canal preparations the endoampullar (receptor) potential (Adc). The postsynaptic activity was monitored by recording the resting and evoked spike activity from the whole ampullar nerve and from single canal afferents. We found that Adc was abolished 5 days after GM administration and that it progressively recovered close to control values 20 days after treatment. These results well fitted with the morphological damage and recovery of hair cells in the crista epithelium. Consistently with the presynaptic damage, the canal afferent activity was abolished 5 days after GM treatment, while the recovery of the background and the evoked afferent activity showed a different sequence. Background activity reappeared 7-8 days after GM treatment and in this period it was not driven by canal mechanical stimulation. In addition, background activity reached control values around 15 days post-treatment, while the evoked activity was detected 9 days after GM treatment and appeared normal close to 20 days. Intracellular recordings from single canal afferents confirmed the above mentioned results and revealed that the EPSP evoked discharge, together with the spike activity, reached normal values close to 20 days post-treatment. The present results show that the frog semicircular canal rapidly and completely recovers its pre -and postsynaptic function following severe ototoxic insult and that during the recovery, the afferent activity at rest becomes functional and mature before the mechano-transduction function

Potassium currents in the hair cells of vestibular epithelium: position-dependent expression of two types of A channels

The complement of voltage-dependent K+ currents was investigated in hair cells of the frog crista ampullaris. The currents were recorded in transversal slices of the peripheral, intermediate and central regions of the crista by applying the whole-cell variant of the patch clamp technique to cells located at different positions in the slices. Voltage-clamp recordings confirmed that cells located in each region have a distinctive complement of K+ channels. A close investigation of the currents in each region revealed that the complement of K+ channels in intermediate and central regions showed no variations among cells, while peripheral hair cells differed in the expression of two classes of A-type currents. These currents showed different kinetics of inactivation as well as steady-state inactivation properties. We called these currents fast IA and slow IA based on their inactivation speed. The magnitude of both currents per unit membrane area exhibited a significant gradient along the transversal axis of the peripheral regions. Fast IA magnitude was maximal in cells located in the external zone of the crista slice and decreased gradually to become very small in the median zone (centre) of the section, while slow IA gradient of magnitude was opposite. A-type currents appear to act as a transient buffer that opposes hair cell depolarisation induced by positive current injections. However, fast IA is partially active at the cell resting potential, while slow IA can be recruited only following large hyperpolarisations. Thus, two types of A currents are differentially distributed in vestibular hair cells and have different roles in shaping the receptor potential

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

11Frogs 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.noneG. Russo;D. Calzi;L. Gioglio;L. Botta;M. R. Polimeni;G. Zucca;M. Martini;D. Contini;R. Fesce;M. L. Rossi;I. PrigioniG., Russo; D., Calzi; L., Gioglio; L., Botta; M. R., Polimeni; G., Zucca; M., Martini; D., Contini; Fesce, RICCARDO GIUSEPPE; M. L., Rossi; I., Prigion

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

Frogs were treated with a single dose of gentamicin sulphate (GM) 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 GM treatment (GMt). At this time the crista epithelium appeared devoid of hair cells. The recovery of the endoampullar potential began around 9 days after GMt and its amplitude progressively increased to reach, after 20 days, values close to those observed in control experiments. The endoampullar potential amplitude appeared to be strictly related to the degree of hair cell regeneration in the crista epithelium. Consistently with the presynaptic damage, the slow generator potential (representing the summed 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 GMt. Conversely, the recovery of the background and evoked afferent activity showed different behaviours. In fact, background spike activity began to be detected 7-8 days after GMt, but it 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 GMt, 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. The absence of any resting and evoked discharge was the most common pattern observed in the early period from 7 to 8 days after GMt. Among the impaled afferents 55% were silent and 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 GMt, 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 GMt, the impaled afferents showed quite normal evoked mEPSP and spike frequencies. The present data indicate that the frog semicircular canal rapidly and completely recovers its pre- and postsynaptic activity following severe ototoxic insult. During the regeneration process, initially the cytoneural junction returns functional and the resting discharge reappears, whereas the mechano-electrical transduction mechanism is restored at later times