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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.

In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.

This study describes the developmental expression of three neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin (NT-4) in the rat auditory brain-stem using immunohistochemistry. At postnatal day 0 (PND 0), neurotrophins expression was virtually absent from all auditory nuclei in the brainstem, even though some positive neurons were observed in the mesencephalic trigeminal nucleus at this age. However, BDNF, NT-3 and NT-4 positive neurons were observed in most brainstem auditory nuclei by PND 6. At the following stages, there was a general increase in the intensity of the neurotrophins immunoreactivity and BDNF labeling was particularly prominent in most cochlear nucleus neurons. A differential pattern of staining emerged in cochlear nucleus subdivisions, with more intense staining present in the ventral part. The superior olivary complex nuclei followed a similar pattern of BDNF staining compared to the cochlear nucleus. In the adult, BDNF heavily labeled most neurons of the superior olivary nuclei and moderately labeled neurons of the inferior colliculus (IC). NT-3 and NT-4 showed a similar pattern of staining in most auditory brainstem nuclei. The first staining was observed by PND 6 in some neuronal cell bodies. NT-3 and NT-4 immunoreactivity increased in the following stages and in the adult moderate labelings were observed in most neurons of the cochlear nucleus, the superior olivary nuclei and the IC. These results show that neurotrophins are expressed 1 week before the onset of hearing and the increase of their expressions correlate with the appearance of sound-evoked activity in the system. The temporal distribution of neurotrophins does not correlate with neuronal birth, axonal outgrowth or the formation of connection in the auditory structures, suggesting a role primarily in the maintenance and/ or modulation of postnatal and adult functions.

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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.

BackgroundPain constitutes the major non motor syndrome in Parkinson's disease (PD) and includes neuropathic pain; however current drug therapies used to alleviate it have only limited efficacy. This is probably due to poor understanding of the mechanisms underlying it.AimsWe investigated a major class of trigeminal neuropathic pain, dynamic mechanical allodynia (DMA), in a rat model of PD and in which a bilateral 6-hydroxy dopamine (6-OHDA) injection was administered to produce a lesion of the nigrostriatal dopaminergic pathway.Results and discussionLesioned animals presented significant DMA in the orofacial area that occurred from 4 days to 5 weeks post-injury. To investigate a segmental implication in the neuropathic pain induced by dopamine depletion, the expression of the isoform gamma of the protein kinase C (PKCg) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) was explored in the medullary dorsal horn (MDH). There was a high increase in PKCg expression in the III and IIi laminae of the MDH of lesioned-animals compared to shams. pERK1/2 expression was also significantly high in the ipsilateral MDH of lesioned rats in response to non-noxious tactile stimulus of the orofacial region. Since pERK1/2 is expressed only in response to nociceptive stimuli in the dorsal spinal horn, the current study demonstrates that non-noxious stimuli evoke allodynic response. Intraperitoneal and intracisternal administrations of bromocriptine, a dopamine 2 receptor (D2R) agonist, significantly decreased DMA compared to control rats injected with saline. These data demonstrate for the first time that nigrostriatal dopaminergic depletion produces trigeminal neuropathic pain that at least involves a segmental mechanism. In addition, bromocriptine was shown to have a remarkable analgesic effect on this neuropathic pain symptom.