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Pimozide is a conventional antipsychotic drug largely used in the therapy for schizophrenia and Tourette's syndrome. Pimozide is assumed to inhibit synaptic transmission at the CNS by acting as a dopaminergic D2 receptor antagonist. Moreover, pimozide has been shown to block voltage-gated Ca2+ and K+ channels in different cells. Despite its widespread clinical use, pimozide can cause several adverse effects, including extrapyramidal symptoms and cardiac arrhythmias. Dizziness and loss of balance are among the most common side effects of pimozide. By using the patch-clamp whole-cell technique, we investigated the effect of pimozide [3 μM] on K+ channels expressed by chicken embryo vestibular type-II hair cells. We found that pimozide slightly blocks a transient outward rectifying A-type K+ current but substantially increases a delayed outward rectifying K+ current. The net result was a significant hyperpolarization of type-II hair cells at rest and a strong reduction of their response to depolarizing stimuli. Our findings are consistent with an inhibitory effect of pimozide on the afferent synaptic transmission by type-II hair cells. Moreover, they provide an additional key to understanding the beneficial/collateral pharmacological effects of pimozide. The finding that pimozide can act as a K+ channel opener provides a new perspective for the use of this drug. [ABSTRACT FROM AUTHOR]

Signal transmission by sensory auditory and vestibular hair cells relies upon Ca2+-dependent exocytosis of glutamate. The Ca2+ current in mammalian inner ear hair cells is predominantly carried through Ca V 1.3 voltage-gated Ca2+ channels. Despite this, Ca V 1.3 deficient mice (Ca V 1.3–/–) are deaf but do not show any obvious vestibular phenotype. Here, we compared the Ca2+ current (I Ca ) in auditory and vestibular hair cells from wild-type and Ca V 1.3–/– mice, to assess whether differences in the size of the residual I Ca could explain, at least in part, the two phenotypes. Using 5 mM extracellular Ca2+ and near-body temperature conditions, we investigated the cochlear primary sensory receptors inner hair cells (IHCs) and both type I and type II hair cells of the semicircular canals. We found that the residual I Ca in both auditory and vestibular hair cells from Ca V 1.3–/– mice was less than 20% (12–19%, depending on the hair cell type and age investigated) compared to controls, indicating a comparable expression of Ca V 1.3 Ca2+ channels in both sensory organs. We also showed that, different from IHCs, type I and type II hair cells from Ca V 1.3–/– mice were able to acquire the adult-like K+ current profile in their basolateral membrane. Intercellular K+ accumulation was still present in Ca V 1.3–/– mice during I K,L activation, suggesting that the K+-based, non-exocytotic, afferent transmission is still functional in these mice. This non-vesicular mechanism might contribute to the apparent normal vestibular functions in Ca V 1.3–/– mice. [ABSTRACT FROM AUTHOR]

Key points Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head., The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L, whose biophysical properties and molecular identity are still largely unknown., In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L., We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme., These results form the basis for a molecular and pharmacological identification of IK,L., Abstract Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential ( VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells. [ABSTRACT FROM AUTHOR]

The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission. [ABSTRACT FROM AUTHOR]

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 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. Detailed investigation of the currents in each region revealed that the complement of K+ channels in intermediate and central regions showed no variations among cells, whereas 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 termed these currents fast IA and slow IA based on their inactivation speed. The magnitude of both currents 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 the gradient of slow IA magnitude was reversed. A-type currents appear to act as a transient buffer that opposes hair cell depolarization 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 hyperpolarizations. Thus, two types of A currents are differentially distributed in vestibular hair cells and have different roles in shaping receptor potential. [ABSTRACT FROM AUTHOR]

The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2-aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1 mm), without affecting mEPSP size or time course. At high concentration (1 mm), 2APB is reported to block the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of group I metabotropic glutamate receptors (RS)-3,5-dihydroxyphenylglycine (DHPG, 0.6 mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1 mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3-sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge. [ABSTRACT FROM AUTHOR]

In the present work we studied the intraregional expression of voltage-dependent Ca2+ and K+ currents in hair cells of frog crista ampullaris. The currents were recorded in situ from sensory cells of the peripheral region, the most populated region of the crista, by using the whole-cell variant of the patch-clamp technique. Voltage-clamp recordings revealed that the calcium current (ICa) and the outward potassium currents of IA, IK, IKCa types and the inward rectifier potassium current of IK1 type exhibited a significant gradient of density (pA/pF) along the region. IA density was maximal in cells located at the beginning of the peripheral region and decreased gradually becoming very small at the opposite end. All the other currents showed an opposite gradient of expression. Current-clamp experiments showed that the voltage behaviour of hair cells changed in relation to cell position. Cells located at the beginning of the peripheral region showed large depolarizations from the resting potential (close to –45 mV) which are consistent with the presence of small IK and IKCa, and an IA largely inactivated at rest. These cells also exhibited slowly developing and large hyperpolarizations that approached passive ones, due to the lack of IK1. In contrast, cells located at the opposite side of the region showed smaller depolarizations and hyperpolarizations from the resting potential (close to –65 mV), due to the presence of large IK and IKCa, and IK1, respectively. The possible role of the intraregional variation of Ca2+ and K+ currents in both hair cell function and afferent discharge properties is discussed. [ABSTRACT FROM AUTHOR]

A correction to the article "Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells," by Elisa Tavazzani and colleagues in the 2014 issue is presented.

Abstract: Ca2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca2+ levels at the apical and basal pole of the cell. [Copyright &y& Elsevier]

Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 ( Eps8 ), a gene involved in actin remodeling, has been shown to cause deafness in mice. While both inner and outer hair cells from Eps8 knockout (KO) mice showed abnormally short stereocilia, inner hair cells (IHCs) also failed to acquire mature-type ion channels. Despite the fact that Eps8 is also expressed in vestibular hair cells, Eps8 KO mice show no vestibular deficits. In the present study we have investigated the properties of vestibular Type I and Type II hair cells in Eps8 -KO mice and compared them to those of cochlear IHCs. In the absence of Eps8, vestibular hair cells show normally long kinocilia, significantly shorter stereocilia and a normal pattern of basolateral voltage-dependent ion channels. We have also found that while vestibular hair cells from Eps8 KO mice show normal voltage responses to injected sinusoidal currents, which were used to mimic the mechanoelectrical transducer current, IHCs lose their ability to synchronize their responses to the stimulus. We conclude that the absence of Eps8 produces a weaker phenotype in vestibular hair cells compared to cochlear IHCs, since it affects the hair bundle morphology but not the basolateral membrane currents. This difference is likely to explain the absence of obvious vestibular dysfunction in Eps8 KO mice. [ABSTRACT FROM AUTHOR]

Abstract: The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca2+ macrocurrent, ICa, and the Ca-dependent K+ current, IKCa. The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (∼34%) of ICa amplitude, without apparently affecting its activation–deactivation kinetics. The IKCa component of the delayed IKD was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca2+ independent fraction of IKD, IKV, and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both ICa and IKD amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of IKD inactivation, although the changes were scaled to the reduced IKD amplitude. From these observations, it is expected that hair cells exposed to gentamicin ‘in vivo’ become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca2+ inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis. [Copyright &y& Elsevier]