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.

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 local expression of proteins involved in handling glutamate may be regulated by the number and activity of synapses in regions of glutamatergic innervation. The systematically varying innervation of inner hair cells (IHCs) of the cochlea provides a model to test this suggestion. IHCs are glutamatergic and form a single row along the cochlear spiral. Along this row the number of afferent fibers terminating on IHCs increases toward the base, reaching a peak and thereafter declining. The afferents are segregated so that higher spontaneous rate fibers terminate on the pillar-cell side of the IHC and lower rate fibers terminate on the modiolar side. Using immunofluorescence and postembedding immunogold labeling, we investigated the distributions of the glutamate-aspartate transporter (GLAST or excitatory amino acid transporter 1), vesicular glutamate transporter (VGLUT1), and the AMPA receptor glutamate receptor 4 (GluR4) along the spiral. Immunofluorescent labeling for GLAST in IHC supporting cells increased in intensity to a peak in the region of 6-9 mm from the apex. Immunogold labeling for GLAST was greater overall in these cells in the 10 mm region than in the 1 mm region and also on the pillar-cell side of the IHC compared with the modiolar side. Immunogold labeling for GluR4 was confined to synaptic sites, represented by puncta in immunofluorescence. The relative numbers of puncta changed with a gradient similar to that of GLAST labeling. VGLUT1 labeling occurred in IHCs but showed no clear cochleotopic gradient. These data suggest that both the density of innervation and the activity levels of glutamatergic synapses may be involved in modulating regional expression of GLAST.

Ains-High levels of collagen type III are biochemically detectable in biopsies of non-uniting fractures, and in the serum of patients suffering from this condition. The aim of this study was to determine whether the expression of collagen type III was limited to fibrous tissue in nonunions, or whether some was present in bone. Methods-Biopsies from normally healing human fractures and non-unions were examined using in situ hybridisation and immunohistochemistry.Results-The mesenchymal cell population, which includes fibroblast and osteoblast precursors, expressed mRNA for collagen type III. However, mature osteoblasts on the surface of woven bone varied profoundly between normally healing fractures (in which they were negative or occasionally weakly positive) and nonunions (in which they were strongly positive). Areas Material and methods PATIENTSSpecimens of human external fracture callus from non-unions were taken from the fracture site of 12 patients with extra-articular nonunited long bone fractures between four and 48 months after fracture. Biopsies from fractures that were healing normally were taken from 15 closed fractures at operations carried out to treat malreduction that had developed during conservative treatment. Biopsies were obtained up to 23 days after fracture. Patients were aged between 18 and 87 years, and were otherwise fit. On subsequent follow up (up to one year), all the fractures classed as healing normally were found to have united conventionally. TISSUE PREPARATIONBiopsy specimens were fixed in 10% neutral buffered formalin, decalcified in 20% ethylenediamine tetra-acetic acid (EDTA), pH 7.2, until decalcification was radiologically complete, embedded in paraffin wax, and sectioned at 7 ,um. IN SITU HYBRIDISATIONThe probes for in situ hybridisation of human procollagen types I and III mRNAs were those prepared, used as described previously,5 and kindly supplied by Dr E Vuorio (University of Turku, Finland). They have minimal homology. Type I: collagen pro 1(I), clone pHCAL1, PvuII-PstI 372 base pair fragment of the 3' C-propeptide region. Type III: collagen pro 1(III), clone pHFS3, PstI-PstI 295 base pair fragment of the 3' telopeptide region.The method for in situ hybridisation has been described previously.'2-16 Briefly, dewaxed sections were rehydrated, pretreated with proteinase K, dehydrated, and air dried; RNAase controls were used. Sections were prehybridised for one hour at 37°C in 50%

Background-Osteoblast phenotypic abnormality, namely the expression of collagen type III, has been shown previously in fracture non-union woven bone. Aims-To investigate osteoblasts from fracture non-unions for evidence of gene expression of non-collagenous bone matrix proteins that have been implicated in mineralisation, namely matrix gla protein (MGP), osteonectin, osteopontin, and osteocalcin. MGP is a consistent component of bone matrix, but there are no reports of osteoblasts in the skeleton expressing the gene for MGP, and the site of synthesis of skeletal MGP (perhaps the liver) has yet to be determined. Methods-Biopsies from normally healing human fractures and non-unions were examined by means of in situ hybridisation, using 35 S labelled probes and autoradiography to disclose levels of gene expression. Results-In normally healing fractures, mature osteoblasts on woven bone were negative for MGP mRNA, but positive for osteonectin, osteopontin, and osteocalcin mRNA molecules. In non-unions, osteoblasts displayed a novel phenotype: they were positive for MGP mRNA, in addition to osteonectin, osteopontin, and osteocalcin mRNA molecules. Conclusions-Mature osteoblasts in slowly healing fractures have an unusual phenotype: they express the gene encoding MGP, which indicates that control of osteoblast gene expression in non-unions is likely to be abnormal. This might be of importance in the pathogenesis of nonuniting human fractures, and is of current interest given the emerging status of MGP as an inhibitor of mineralisation. (J Clin Pathol: Mol Pathol 1999;52:92-96)