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. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K+ exiting the hair cell. To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K+] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K+] was primarily driven by GK,L, the low-voltage-activated, non-inactivating K+ conductance specifically expressed by Type I hair cells. Simple diffusion of K+ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K+]. Calyx recordings were consistent with K+ leaving the synaptic cleft through postsynaptic voltage-gated K+ channels involving KV1 and KV7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K+.

Physiological recordings from the zebrafish lateral line.

During sensory transduction, external physical stimuli are translated into an internal biological signal. In vertebrates, hair cells are specialized mechanosensory receptors that transduce sound, gravitational forces, and head movements into electrical signals that are transmitted with remarkable precision and efficiency to afferent neurons. Hair cells have a conserved structure between species and are also found in the lateral line system of fish, including zebrafish, which serve as an ideal animal model to study sensory transmission in vivo. In this chapter, we describe the methods required to investigate the biophysical properties underlying mechanosensation in the lateral line of the zebrafish in vivo from microphonic potentials and single hair cell patch-clamp recordings to single afferent neuron recordings. These techniques provide real-time measurements of hair-cell transduction and transmission following delivery of controlled and defined stimuli and their combined use on the intact zebrafish provides a powerful platform to investigate sensory encoding in vivo.

Hair cells--beyond the transducer.

OVERVIEW: This review considers the "tween twixt and twain" of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage. Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility. Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function.

A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles.

A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.

Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations.

Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB), on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels.

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. I(A) 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 I(K) and I(KCa), and an I(A) largely inactivated at rest. These cells also exhibited slowly developing and large hyperpolarizations that approached passive ones, due to the lack of I(K1). 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 I(K) and I(KCa), and I(K1), respectively. The possible role of the intraregional variation of Ca2+ and K+ currents in both hair cell function and afferent discharge properties is discussed.

FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel.

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.

Developmental expression of the potassium current IK,n contributes to maturation of mouse outer hair cells.

1. The expression of K+ currents in mouse outer hair cells (OHCs) was investigated as a function of developmental age between postnatal day (P) 0 and P26, using whole-cell patch clamp. 2. During the first postnatal week, a slow outward K+ current (IK,neo) was expressed by all OHCs from the apical coil of the cochlea. The amplitude of this current increased greatly between P0 and P6. Then, at the beginning of the second postnatal week, IK,neo decreased. At the same time, from P8 onwards, IK,n, a K+ current characteristic of mature OHCs, was rapidly expressed. 3. The expression of IK,n coincided with the onset of electromotility of the cell body of the OHCs, which could also be detected from P8 onwards and increased substantially in size thereafter. 4. IK,n was reversibly blocked by linopirdine, an inhibitor of members of the KCNQ family of K+ channels, with a KD of 0.7 microM. In the cochlea, KCNQ4 is only expressed in OHCs and is responsible for a form of non-syndromic autosomal dominant deafness. Linopirdine had no effect on other OHC K+ currents at concentrations up to 200 microM. We conclude that ion channels underlying IK,n contain the KCNQ4 subunit. 5. In current clamp, depolarizing current injections from the resting potential triggered action potentials in OHCs during the first postnatal week. Thereafter, more rapid and graded voltage responses occurred from more negative resting potentials. Thus, OHCs mature rapidly from P8 onwards, and IK,n contributes to this maturation.

Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells.

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.

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)). I(K,c) was recruited at potentials more positive than -60 mV and showed a slow activation having a time constant (tau(a)) ranging on average from 12 ms at 40 mV to 32 ms at -20 mV. This current inactivated slowly with two voltage-independent time constants (ta(d1) and tau(d2) were 300 ms and 4 s respectively) and more than 80% of the channels were in an inactivated state at the cell resting potential. I(K,b) was also recruited at potentials more positive than -60 mV, but in contrast to I(K,c), it activated more rapidly (tau(a) ranged on average from 1 ms at 40 mV to 4.5 ms at -20 mV) and it did not exhibit any inactivation process. Current clamp experiments revealed that I(K,b), at variance with I(K,c), contributes to the cell resting potential and represents the main repolarizing current when sensory cells are depolarized from rest. I(K,c) could have a role in hair cells when they are depolarized after hyperpolarizing stimuli, a condition that removes channel inactivation.

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+. I(K1) was found in virtually all cylindrical hair cells of the central region and in club-shaped cells located in the halves of the peripheral regions closest to the centre of the crista. Pear-shaped cells of the intermediate regions showed no inward rectification. The I(K1) density (pA/pF) varied along the crista depending on cell position, being maximal in cells located in the middle of the central region and decreased towards its ends. In the peripheral regions, the gradient of I(K1) increased towards the centre of the crista. Current clamp experiments showed that sensory cells having larger I(K1) constantly exhibited more negative resting potentials and required more depolarizing current to elicit an active response than cells having small or no I(K1).

Transient expression of an inwardly rectifying potassium conductance in developing inner and outer hair cells along the mouse cochlea.

Inwardly rectifying K+ currents in inner and outer hair cells (IHCs, OHCs) were studied during post-natal development of the mouse cochlea. Hyperpolarizing steps from a holding potential of -64 mV induced a rapidly activating current in both cell types. This current showed strong inward rectification around the K+ equilibrium potential and, at potentials negative to -130 mV, partial inactivation. The activation range varied with extracellular K+ concentration. External application of Ba2+ and Cs+ reversibly blocked the elicited current. The results are consistent with the presence of an IK1-type inwardly rectifying potassium conductance in these cells. The maximum current was 60% larger in IHCs than in OHCs. In OHCs, but not IHCs, the amplitude of IK1 varied significantly with the cells' position along the cochlea. IK1 was maximal in cells located in the most basal region of the cochlea and its amplitude decreased in the apical coil. IK1 disappeared upon functional maturation: in OHCs at the end of the first postnatal week, and in IHCs at the onset of auditory function 12 days after birth. The current is active at the resting potential of the cells and plays a role in regulating the spiking behaviour characteristic of developing hair cells.

A missense mutation in myosin VIIA prevents aminoglycoside accumulation in early postnatal cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the myosin VIIA molecule we show that this myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for myosin VIIA in the process of aminoglycoside accumulation are discussed.

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. No reaction products were detectable on the basolateral membranes and in cytoplasmatic organelles. Supporting cells of the crista showed a quite variable Ca-ATPase reaction on microvilli and on basolateral membranes. The presence of an evident reactivity on the stereocilia is consistent with the existence of an apical calcium microdomain involved in the mechano-transduction process and supports the current view that calcium ions enter the stereocilia during natural stimulation. On the other hand, the lack of an observable reactivity on the basolateral membrane of hair cells suggests that in semicircular canals other mechanisms of active transport of calcium ions across the plasma membrane, such as Na-Ca exchange, may be involved in homeostasis of the ion.

Somatotopic nucleocortical projections to the multiple somatosensory cerebellar maps.

The cerebellum is organized in a series of parasagittal compartments: in C1-C3 and C2 compartments Purkinje cells receive climbing fibre afferents from the rostral part of the accessory olives, and project their axon to the nucleus interpositus anterior and posterior, respectively. Within these compartments electrophysiological studies have shown that the cutaneous input carried by climbing fibre afferents is topographically organized so as to design a map of peripheral body districts. The body map is replicated over the anterior lobe-pars intermedia and the paramedian lobule, and anatomical studies have indicated that the replication is partly due to the axonal branching of olivocerebellar neurons. The aim of this study was to analyse the presence of a somatotopic organization and of a branching pattern in the nucleocortical projections, in relation to the replicated body maps within C1-C3 and C2 compartments. By using double retrograde neuronal tracing we explored, in the cat, the topographic distribution of single- and double-labelled cells in the interposed nuclear subdivisions, after tracer injections into forelimb or hindlimb regions of the anterior lobe-pars intermedia, paramedian lobule and hemisphere (medial crus II). Most of the nucleocortical neurons were found in ipsilateral nucleus interpositus posterior, with smaller numbers in the ipsilateral nucleus interpositus anterior. Nucleocortical neurons projecting to forelimb- or hindlimb-related areas are completely segregated, the forelimb neurons being located laterally and the hindlimb neurons medially in the nucleus interpositus posterior. Within their respective domains both the forelimb and hindlimb populations projecting to the anterior lobe-pars intermedia are partly segregated from those projecting to the paramedian lobule, in that the two populations are slightly shifted along the dorsoventral axis of the nucleus. Although mostly different, some of the cells are common to the two forelimb populations, since they send axonal branches to the homologous areas of the anterior lobe and paramedian lobule. Contralateral fastigial or interposed nucleocortical projections are restricted to the anterior lobe-pars intermedia, and their neurons of origin are different from those that project to the ipsilateral cerebellar cortex: i.e. they are not a bilateral, but a separate contralateral component.

Neuropeptide Y (NPY) expression is up-regulated in the rat inferior olive during development.

The aim of this study was to analyse the developmental expression of the neuropeptide Y (NPY) in the rat inferior olivary (IO) complex by immunoperoxidase and immunofluorescence techniques. The spatial distribution of NPY-immunoreactivity (IR) did not vary during development, whereas NPY-IR intensity levels varied significantly. The peak of NPY-IR expression occurred during the second postnatal week, but differed in intensity in individual IO subnuclei, the highest levels being present in the dorsal fold of the dorsal accessory olive and in the ventro-lateral outgrowth. In the adult, NPY-IR could only be rescued in colchicine pretreated animals, but its distribution overlapped the one found during development. These findings show that NPY-IR is transiently up- regulated, during development, in specific compartments of the IO complex, and that the peptide is rescued in the same specific olivocerebellar compartments in the adult. These observations are here taken to support the hypothesis that NPY may exert different trophic-differentiating and/or neuromodulatory roles during development, when its expression is transiently up-regulated, or at adult stages, when it can be rescued, according to the different biological contexts.

Neuronal and glial localization of the GABA transporter GAT-1 in the cerebellar cortex.

THE distribution and neuronal or glial localization of GAT-1, a high affinity GABA transporter, in the cerebellar cortex was analysed by means of double-label immunofluorescence experiments with GAT-1 combined with calbindin D, synaptophysin, or GFAP antibodies. In the Purkinje cell (Pc) layer, prominent synaptic GAT-1-immunoreactivity (IR) was found in the axon terminals of basket cells surrounding the Pc axon hillock. GAT-1-IR was also found in neuronal and glial processes ensheathing Pc somata and dendrites. Numerous immunoreactive fibres and puncta originating from basket and stellate cells, or from Golgi cells, were also detected in the molecular or granular layer, respectively. These observations suggest that GAT-1 is involved in the termination of the action of GABA at the inhibitory synapses of all cerebellar interneurones, primarily of basket cell terminals at the Pc axon hillock. GAT-1 in the astroglial processes presumably plays the additional role of regulating the extracellular concentrations of GABA.

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. Ik showed clear time-dependent inactivation over a period of some seconds, but the current did not completely inactivate even after 30 s depolarizing pulses. Another interesting finding was that inactivation could be well fitted by the sum of two exponentials: at 20 mV depolarization the fast time constant was 291.3 ms and the slow time constant was 2662.3 ms. In addition, an analysis of the steady-state inactivation process of Ik revealed that the inactivation curve was incomplete showing a non-inactivating current at potentials more positive than -50 mV. These results suggested that Ik in hair cells of frog crista ampullaris is composed of more than one component: by at least one inactivating and one non-inactivating component. The possible role of these components in hair cell excitability is discussed.

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). IK and IK(Ca) exhibited similar magnitude and accounted for most of the membrane cell conductance. The same experimental protocol applied to cells from the intermediate regions showed the presence of a large and sustained IK(Ca) which represented 95% of the total outward current. In this region IA was absent. The present results demonstrated that pear-shaped hair cells located in two discrete regions of frog crista ampullaris exhibit a different complement of voltage-dependent conductances, suggesting that they can play a different role in processing the natural stimulus.

Comparison of three histochemical methods for assaying lactate dehydrogenase in liver.

The mean activity of lactate dehydrogenase (LDH) in hepatocytes near the central vein region of unfixed sections of mouse liver was determined and compared with 3 different histochemical methods: conventional method, polyvinyl alcohol (PVA) method, and gel film method. An image analysis system was used for measuring the enzyme activity in single hepatocytes. The mean activities were approximately 1.4 and 2.7 times higher with the PVA and a gel film techniques respectively than with conventional aqueous media. The highest activity of LDH was obtained with gel media; this can be explained by the lowest diffusion late of this soluble cytoplasmic enzyme from the secretion into the medium. In the conventional technique, the apparent activity was found to be about 16% lower when sections were incubated vertically in a large volume of medium than when they were incubated horizontally in a small volume of medium.