K Accumulation and Clearance in the Calyx Synaptic Cleft of Type I Mouse Vestibular Hair Cells.

Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors.

An allosteric gating model recapitulates the biophysical properties of I expressed in mouse vestibular type I hair cells.

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, I , 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 I .

Distinct roles of Eps8 in the maturation of cochlear and vestibular hair cells.

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.

Corrigendum: Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

[This corrects the article on p. 428 in vol. 8, PMID: 25565962.

Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

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.

Intercellular K⁺ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx.

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells.

Acute effects of gentamicin on the ionic currents of semicircular canal hair cells in the frog.

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 Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (∼34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics.

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.

Ionic currents in hair cells dissociated from frog semicircular canals after preconditioning under microgravity conditions.

The effects of microgravity on the biophysical properties of frog labyrinthine hair cells have been examined by analyzing calcium and potassium currents in isolated cells by the patch-clamp technique. The entire, anesthetized frog was exposed to vector-free gravity in a random positioning machine (RPM) and the functional modification induced on single hair cells, dissected from the crista ampullaris, were subsequently studied in vitro. The major targets of microgravity exposure were the calcium/potassium current system and the kinetic mechanism of the fast transient potassium current, I(A).

Plasma membrane Ca2+-ATPase isoforms in frog crista ampullaris: identification of PMCA1 and PMCA2 specific splice variants.

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.

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 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.

IP3 receptor in the hair cells of frog semicircular canal and its possible functional role.

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.

Presynaptic calcium stores modulate afferent release in vestibular hair cells.

Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter.

Nature and expression of dihydropyridine-sensitive and -insensitive calcium currents in hair cells of frog semicircular canals.

Ca(2+) currents in hair cells of the frog crista ampullaris were studied using the whole-cell patch-clamp technique. Currents were recorded in situ from hair cells in peripheral, intermediate and central regions of the sensory epithelium. Two types of Ca(2+) currents were found: a partially inactivating current that was expressed by nearly all central cells and by about 65% of intermediate and peripheral cells, and a sustained current expressed by the remaining cell population.

Ecto-ATPase activity sites in vestibular tissues: an ultracytochemical study in frog semicircular canals.

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction.

Gradients of expression of calcium and potassium currents in frog crista ampullaris.

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 (I(Ca)) and the outward potassium currents of I(A), I(K) I(KCa) types and the inward rectifier potassium current of I(K1) type exhibited a significant gradient of density (pA/pF) along the region.

Inactivating and non-activating delayed rectifier K+ currents in hair cells of frog crista ampullaris.

The possible presence of different types of delayed rectifier K+ current (I(K)) was studied in vestibular hair cells of frog semicircular canals. Experiments were performed in thin slice preparations of the whole crista ampullaris and recordings were made using the whole-cell patch-clamp technique. We found that an apparent homogeneous I(K), isolated from the other K+ currents, could be pharmacologically separated into two complementary components: a capsaicin-sensitive current (I(Kc)) and a barium-sensitive current (I(K,b)).

Position-dependent expression of inwardly rectifying K+ currents by hair cells of frog semicircular canals.

The identity and the expression of inwardly rectifying ionic currents were studied using the whole-cell variant of the patch-clamp technique in frog semicircular canal hair cells in situ. The currents were examined in club-, cylindrical- and pear-shaped sensory cells located in three discrete regions of the crista. A unique current of I(K1) type was distinguished based on its K+ selectivity, rapid monoexponential activation, dependence of activation on external K+ and blockade by Ba2+ and Cs+.

Localization of Ca-ATPase in frog crista ampullaris.

The distribution of Ca-ATPase in frog crista ampullaris was mapped ultracytochemically by using a one-step lead citrate reaction. Electron-dense precipitates, as an expression of Ca-ATPase activity, were observed on the surface of stereocilia and on the apical membrane surrounding the cuticular plate of hair cells. Sensory cells of the isthmus region showed more reactivity than those of the peripheral regions of the crista.

Inactivation of delayed rectifier K+ current in semicircular canal hair cells.

Some properties of the inactivation process of delayed rectifier K+ current (Ik) were investigated in vestibular hair cells of the central region of frog crista ampullaris. These cells were chosen since they exhibited a very large Ik. Experiments were performed on thin slices of sensory epithelium using the whole-cell variant of the patch-clamp technique.

Potassium currents of pear-shaped hair cells in relation to their location in frog crista ampullaris.

Voltage-dependent K+ currents in pear-shaped hair cells of the frog crista ampullaris were investigated in thin slice preparations using the whole-cell variant of the patch-clamp technique. Microscopy observation revealed that pear-shaped cells are located in intermediate and peripheral regions of the crista, whereas they are absent in the central region. Voltage-clamp recordings in cells from the peripheral regions revealed that the total outward K+ current could be separated pharmacologically into three distinct components: a A-type K+ current (IA); an inactivating calcium-activated K+ current (IK(Ca)) and a delayed rectifier K+ current (IK).

Glutamate excitatory effects on ampullar receptors of the frog.

The action of glutamate on frog ampullar receptors was investigated to assess the potential role of this excitatory amino acid as an afferent transmitter in the hair cell system. Intracellular recordings from single afferent units in the isolated labyrinth revealed that glutamate and the glutamate receptor agonists, N-methyl-D-aspartic acid, quisqualic acid and kainic acid increase dose-dependently the frequency of the resting afferent discharge of EPSPs and spikes and produce long lasting depolarizations. After blocking synaptic transmission by using 5 mM Co(2+), the same compounds elicited only depolarizations of amplitude comparable to those observed in normal saline.

Morphological features of different regions in frog crista ampullaris (Rana esculenta).

The cellular organization of different regions of the crista epithelium from the frog posterior semicircular canal was studied by light, transmission and scanning microscopy. The sensory epithelium consists of hair cells surrounded by supporting cells and basal cells located close to the basement membrane. Three types of hair cells, namely club-like, cylindrical and pear-like cells differentially distributed along the crista could be recognized on the basis of their shape.

Isolation of A-type K+ current in hair cells of the frog crista ampullaris.

Different procedures to isolate the K+ A-type current (IA) from other membrane currents were tested on the complex inactivating outward K+ current generated in hair cells from the peripheral regions of the frog crista ampullaris. Experiments were performed in thin slices of epithelium using the whole-cell configuration of the patch-clamp technique. The conventional conditioning voltage protocol did not allow a satisfactory isolation of IA, due to the presence of other K+ currents showing overlapping steady-state inactivation properties.