David N. Furness

Cross-links between stereocilia in the guinea pig cochlea

Cross-links between stereocilia on guinea pig cochlear hair cells have been examined using high resolution scanning (SEM) and transmission electron microscopy (TEM), confirming recent descriptions of these structures. Links from the tips of shorter stereocilia to the sides of the adjacent taller stereocilia (upward-pointing links), between stereocilia of the same row (side-to-side links) and between adjacent rows (row-to-row links), have been observed on inner and outer hair cells. These links have been seen in material fixed using (1) glutaraldehyde only, (2) glutaraldehyde/osmium and (3) glutaraldehyde/osmium/thiocarbohydrazide (a technique which makes gold coating unnecessary). Upward-pointing links were seen less frequently, and the surfaces of stereocilia and microvilli were smoother after fixation (3) compared with fixations (1) and (2) in which they were usually roughened in appearance. In TEM, side-to-side and row-to-row links form a regular lattice between stereocilia, and consist of a number of strands. Upward-pointing links consist of a single strand, the ends of which are associated with electron-dense material. This lies between the stereociliary membrane and the actin filament bundle, at the tip of the shorter stereocilium and the side of the taller stereocilium.

A QUANTITATIVE ASSESSMENT OF GLUTAMATE UPTAKE INTO HIPPOCAMPAL SYNAPTIC TERMINALS AND ASTROCYTES : NEW INSIGHTS INTO A NEURONAL ROLE FOR EXCITATORY AMINO ACID TRANSPORTER 2 (EAAT2)

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

Localization of the glutamate–aspartate transporter, GLAST, in rat taste buds

A number of putative neurotransmitter substances have been found in vertebrate taste buds. Amongst these glutamate has been localized in fibres innervating the buds and uptake of glutamate has been shown to occur into receptor cells. It is therefore possible that, in common with other sensory systems, glutamate is a neurotransmitter in taste buds. In the inner ear and retina of mammals, the membranes of supporting cells have been shown to contain the glial glutamate transporter GLAST. In the brain, this protein is involved in glutamate re‐uptake into glial cells where the glutamate is converted into glutamine for recycling into glutamatergic terminals. In this study, the presence of GLAST has been investigated in taste buds in the rat vallate papilla and its distribution compared with that of glutamine to determine whether there are cells in this system that play a glia‐like role in glutamate handling. Immunofluorescent labelling showed that a subset of cells in the taste bud contains GLAST. Immunogold labelling indicated that it occurs in the plasma membranes of supporting cells, especially on the fine cytoplasmic processes of dark cells towards the basal region of the bud. A protein of molecular mass similar to that of cerebellar GLAST was detected in immunoblots of excised papillae. Double labelling and semiquantitative analysis of glutamine and GLAST immunoreactivity showed that the GLAST‐positive cells have a higher level of cytoplasmic glutamine than the adjacent cells. It is proposed that these GLAST‐positive cells play a glia‐like role in the uptake of glutamate following its release at synapses within the taste bud although the precise location of the latter remains uncertain. The GLAST‐positive cells may also be involved in its subsequent conversion to glutamine in a glutamate/glutamine cycle similar to that described in the brain.

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm−3), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-d-aspartate (4-methoxy-7-nitroindolinyl-d-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and d-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca2+ dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca2+ sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca2+ dependence of immature spiking cells. However, the nature of the Ca2+ sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca2+ dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca2+ dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca2+ sensitivity of vesicle fusion at hair cell synapses.

Auditory Hair Cell-Afferent Fiber Synapses Are Specialized to Operate at Their Best Frequencies

Auditory afferent fiber activity is driven by high-fidelity information transfer from the sensory hair cell. Presynaptic specializations, posited to maintain fidelity, are investigated at synapses with characteristic frequencies of 120 Hz and 320 Hz. Morphological data indicate that high-frequency cells have more synapses and higher vesicle density near dense bodies (DBs). Tracking vesicular release via capacitance changes identified three overlapping kinetic components of release corresponding to morphologically identified vesicle pools. High-frequency cells released faster; however, when normalized to release site number, low-frequency cells released faster, likely due to a greater Ca2+ load per synapse. The Ca(2+)-dependence of release was nonsaturating and independent of frequency, suggesting that release, not refilling, was rate limiting. A model of release derived from vesicle equilibration between morphologically defined pools reproduced the capacitance data, supporting a critical role in vesicle trafficking for DBs. The model suggests that presynaptic specializations enable synapses to operate most efficiently at their characteristic frequencies.

IMMUNOCYTOCHEMICAL LOCALIZATION OF A HIGH-AFFINITY GLUTAMATE-ASPARTATE TRANSPORTER, GLAST, IN THE RAT AND GUINEA-PIG COCHLEA

Glutamate transporters play an important role in the reuptake of glutamate after its release from glutamatergic synapses. Four such transporters have so far been cloned from the rat brain. One, the glutamate‐aspartate transporter GLAST, has been detected in the mammalian cochlea, in which the principal afferent synapse of the auditory nerve, between the inner hair cells and neurites of type I spiral ganglion neurons, has been suggested to be glutamatergic. The distribution of GLAST was therefore investigated to provide clues to the handling of glutamate in the cochlea. This was studied using light and electron microscopic immunocytochemistry in rats and guinea pigs with antibodies raised against synthetic peptides based on the sequence for GLAST. Significant immunoreactivity was found in the myelin sheath formed by satellite cells surrounding the type I spiral ganglion neurons, and along the plasma membranes of supporting cells around the inner hair cells; other cells in both locations were only weakly labelled, if at all. The absence of substantial numbers of synapses in the spiral ganglion suggests that GLAST is unlikely to be associated with the uptake of synaptic glutamate after release in this region. lmmunoreactivity associated with the inner hair cells is consistent with the utilization of glutamate at the afferent synapse.

Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells

Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cells mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure

The distribution of tubulin has been investigated in surface preparations of the guinea pig organ of Corti using indirect immunofluorescence microscopy. Two different monoclonal antibodies to tubulin produce similar distinct patterns of labelling in hair cells. Labelling is greater in inner hair cells than outer hair cells. It occurs in rings around the cell apex, and in a meshwork below and channels through, the cuticular plate. In outer hair cells from the apical region of the cochlea, labelling occurs around the location of a basalward protrusion of the cuticular plate. These patterns correlate with the location of microtubules observed using transmission electron microscopy. A large patch of labelling occurs on the strial side of the cell corresponding to the largest channel through the cuticular plate and the kinociliary basal body. Strands of labelling are seen running parallel to the long axis of the cell between the subcuticular and synaptic region. Many more of these strands are seen in the inner hair cell than the outer hair cell and may correspond to tracks of microtubules transporting neurotransmitter vesicles or other organelles. In outer hair cells, intense labelling and many microtubules are seen in the subnuclear region. The possible roles of the different microtubule arrangements are discussed.