Intracochlear perfusion of pneumolysin, a pneumococcal protein, rapidly abolishes auditory potentials in the Guinea pig cochlea.

OBJECTIVE: Bacterial meningitis and chronic suppurative otitis media caused by Streptococcus pneumoniae are associated with considerable otological morbidity. Specifically, sensorineural hearing loss is a permanent sequela in a third of those who contract pneumococcal meningitis. Pneumolysin, a pneumococcal protein, has been implicated as one of the main virulence/cytotoxic factors. Its pathogenicity is intimately dependent on an ability to form transmembrane pores on binding with cholesterol in target tissues. MATERIAL AND METHODS: We perfused wild-type pneumolysin, at a number of different concentrations, into the guinea pig cochlea and used electrocochleography to characterize the effects of this cytolytic exotoxin in the organ of Corti. RESULTS: Intracochlear perfusion of pneumolysin (10 microg/50 microl) reduced the compound action potential of the auditory nerve within seconds. The cochlear microphonics (f1=8 kHz, f2=9.68 kHz) and their distortion product (2f1-f2) were also reduced, albeit in a slightly less dramatic fashion. At lower concentrations (1 microg/50 microl), a selective and earlier effect on inner hair cells was observed. CONCLUSIONS: These results clearly show that significant ototoxicity ensues when sensory cells of the organ of Corti are exposed to pneumolysin (and complete cochlear death when the concentration is high enough). Toxicity is dose-dependent and appears to be site-sensitive. This may have implications for any possible future protective strategies against pneumococcal disease in the ear.

Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells.

Ryanodine receptors (RyRs) are known to contribute to the regulation of free cytosolic calcium concentration. This family of intracellular calcium channels plays a significant role in calcium-induced-calcium-release (CICR), and have been implicated in calcium-dependent processes requiring exquisite spatio-temporal regulation. In order to characterize the importance of these intracellular calcium channels in cochlear physiology, we perfused the guinea pig cochlea with antagonistic concentrations of ryanodine. The distortion products of the cochlear microphonic and the compound action potential of the auditory nerve were reversibly inhibited by ryanodine (IC(50)=27.3 microm, Hill coefficient=1.9), indicating an action at the cochlear amplifier. Single auditory nerve fibre recordings showed that ryanodine slightly increased spontaneous firing rates by 22%, suggesting an excitatory effect of ryanodine. This paradoxical effect could be explained by an inhibitory action of ryanodine on presynaptic BK channels of inner hair cells (IHC). Indeed, perfusing iberiotoxin also increased the spontaneous firing activity of the auditory nerve fibres. Furthermore, whole-cell patch-clamp recordings demonstrated that ryanodine inhibits BK currents at the IHC level. Conversely, immunohistochemistry demonstrated a strong expression of RyR in IHCs and, more particularly, below the cuticular plate where membranous BK channels are highly expressed. Overall, the study demonstrated a key role for RyR and CICR in signal transduction at the IHCs. We therefore propose that coupled RyR--BK channels act to suppress the fast neurotransmission in IHCs.

Effects of exposure of the ear to GSM microwaves: in vivo and in vitro experimental studies.

The effects of mobile phone (GSM) microwaves on the ears of guinea pigs were investigated in two in vivo experiments and one in vitro experiment. In the first experiment, three groups of eight guinea pigs had their left ear exposed for 1 h/day, 5 days/week, for 2 months, to GSM microwaves (900 MHz. GSM modulated) at specific absorption rates (SARs) of 1, 2 and 4 W/kg respectively, and a fourth group was sham-exposed. Distortion-product otoacoustic emissions (DPOAEs) were measured for each ear before exposure, at the end of the 2-month exposure period, and 2 months later. In the second experiment, the same protocol was applied to eight sham-exposed and 16 exposed guinea pigs at 4W/kg, but the auditory brainstem response (ABR) thresholds were monitored. Repeated-measures ANOVA showed no difference in DPOAE amplitudes or in ABR thresholds between the exposed and non-exposed ears and between the sham-exposed and exposed groups In the course of the second experiment, acute effects were also investigated by measuring once, in all animals, ABR thresholds just before and just after the 1-h exposure: no statistically significant difference was observed. In vitro, the two organs of Corti (OCs) of newborn rats (n=15) were isolated and placed in culture. For each animal, one OC was exposed for 24-48 h to 1 W/kg GSM microwaves, and the other was sham-exposed. After 2-3 days of culture, all OCs were observed under light microscopy. They all appeared normal to naive observers at this stage of development. These results provided no evidence that microwave radiation, at the levels produced by mobile phones, caused damage to the inner ear or the auditory pathways in our experimental animals.

Contribution of BK Ca2+-activated K+ channels to auditory neurotransmission in the Guinea pig cochlea.

Large-conductance calcium-activated potassium (BK) channels are known to play a prominent role in the hair cell function of lower vertebrates where these channels determine electrical tuning and regulation of neurotransmitter release. Very little is known, by contrast, about the role of BK channels in the mammalian cochlea. In the current study, we perfused specific toxins in the guinea pig cochlea to characterize the role of BK channels in cochlear neurotransmission. Intracochlear perfusion of charybdotoxin (ChTX) or iberiotoxin (IbTX) reversibly reduced the compound action potential (CAP) of the auditory nerve within minutes. The cochlear microphonics (CM at f1 = 8 kHz and f2 = 9.68 kHz) and their distortion product (DPCM at 2f1-f2) were essentially not affected, suggesting that the BK specific toxins do not alter the active cochlear amplification at the outer hair cells (OHCs). We also tested the effects of these toxins on the whole cell voltage-dependent membrane current of isolated guinea pig inner hair cells (IHCs). ChTX and IbTX reversibly reduced a fast outward current (activating above -40 mV, peaking at 0 mV with a mean activation time constant tau ranging between 0.5 and 1 ms). A similar block of a fast outward current was also observed with the extracellular application of barium ions, which we believe permeate through Ca2+ channels and block BK channels. In situ hybridization of Slo antisense riboprobes and immunocytochemistry demonstrated a strong expression of BK channels in IHCs and spiral ganglion and to a lesser extent in OHCs. Overall, our results clearly revealed the importance of BK channels in mammalian cochlear neurotransmission and demonstrated that at the presynaptic level, fast BK channels are a significant component of the repolarizing current of IHCs.