Marlene Shero

Endocytosis of aminoglycoside antibiotics in sensory hair cells

Immuno-gold electron microscopy was used to assess the uptake pathways of aminoglycoside antibiotic kanamycin (KM) in sensory hair cells. Accumulation of gold particles was evident on the plasma membrane as well as in large smooth vesicles beneath the apical surfaces of hair cells 12 h after a systemic administration of KM. Immuno-gold was exclusively localized in the vesicles 27 h post-injection. Cationic ferritin, a membrane-bound insoluble marker, was colocalized with KM in the vesicle structures after their simultaneous in vitro application. These results strongly suggest that KM is taken up into sensory hair cells via receptor-mediated endocytosis at their apical surfaces. In addition, the profound time lag between KM uptake and hair cell death suggests involvement of targeting mechanisms in cytotoxic signalling pathways of the drugs.

Lysosomal targeting and accumulation of aminoglycoside antibiotics in sensory hair cells.

Our recent study demonstrated that aminoglycoside antibiotics are taken up into sensory hair cells of the inner ear by receptor-mediated endocytosis (E. Hashino, M. Shero, Endocytosis of aminoglycoside antibiotics in sensory hair cells, Brain Res. 704 (1995) 135-140). To elucidate the intracellular trafficking pathway of aminoglycosides following endocytotic uptake, we administered kanamycin to neonatal chicks for 1 or 5 days (400 mg/kg/day) and determined the location of kanamycin within the hair cells at various time points using immunogold electron microscopy. Quantitative and qualitative analysis of immunogold staining revealed that: (1) kanamycin was primarily localized in vesicles beneath the cuticular plate 27 h postinjection; (2) the number of vesicles per hair cell and the number of gold particles per vesicle increased over time; (3) individual vesicles tended to increase in size over time, presumably due to aggregation of smaller vesicles; and (4) in pathological hair cells, immunogold was dispersed throughout the entire subcellular region. Light microscopic observations of the basilar papilla stained with the same antibody confirmed the temporal changes in the kanamycin distribution. Moreover, results obtained from acid phosphatase cytochemistry indicated that vesicles accumulating kanamycin were mainly lysosomes. These results suggest that internalized aminoglycosides are transported via vesicular traffic into lysosomes where they accumulate over time and lead to disruption of lysosomes. The time of diffusion of kanamycin was closely related to the time of cell death, suggesting that lysosomal rupture could be a direct trigger for the hair cell degeneration.

Cochlear frequency-place map in adult chickens: Intracellular biocytin labeling

A cochlear frequency-place map was developed for adult chickens by labeling cochlear ganglion neurons with biocytin and correlating the location of each labeled fiber along the basilar papilla with the characteristic frequency of the units tuning curve. Labeled fibers showed little or no branching within the sensory epithelium and most fibers appeared to terminate on a single hair cell along the neural side of the basilar papilla. The CFs of the labeled neurons ranged from 353 Hz to 3145 Hz and the location of the labeled neurons ranged from 30.1% to 74.4% of the total distance from the apex of the papilla. CFs increased in an orderly manner from the apex towards the base of the papilla. The cochlear frequency map for adult chickens was similar to that estimated from previous cochlear lesion studies carried out on 30 day old chicks, although the predicted frequencies in the adults were slightly higher in some regions of the basilar papilla than in 30 day old animals. However, previous maps developed in young animals (< or = 21 days) using lesion or labeling data predict significantly lower frequencies for a given location than in adult animals particularly in the basal half of the cochlea.

Tuning, spontaneous activity and tonotopic map in chicken cochlear ganglion neurons following sound-induced hair cell loss and regeneration.

Adult chickens were exposed for 48 h to a 525 Hz, 120 dB SPL tone that destroyed the hair cells and tectorial membrane in a crescent-shaped patch along the abneural edge of the basilar papilla. Single-unit recordings were obtained from cochlear ganglion neurons 0-1, 5, 14 and 28 days post-exposure to determine what effect the cochlear lesion had on neural discharge patterns and if the discharge patterns fully recovered. Immediately after exposure, the tuning curves were extremely broad and CF thresholds were elevated by 30-40 dB. In addition, the average spontaneous rate and percentage of neurons with interspike interval histograms with preferred intervals were greatly reduced. Tuning curves and spontaneous activity started to recover by 5 days post-exposure; however, some W-shaped tuning curves with two distinct tips and a hypersensitive tail were observed at this time. W-shaped tuning curves disappeared and spontaneous activity recovered to normal levels 14-28 days post-exposure. However, the CF thresholds of the most sensitive neurons were still slightly elevated, tuning curve slopes below CF were shallower than normal, and thresholds in the low-frequency tail of the tuning curves were often hypersensitive. These functional deficits were most closely associated with residual damage to the upper fibrous layer of the tectorial membrane. To determine if the cochlear frequency-place map was altered by the cochlear lesion, four physiologically characterized neurons were labeled with biocytin at 5 days post-exposure. The CFs of the labeled neurons were consistent with the normal frequency-place map (Chen et al. (1994) Hearing Research 81, 130-136) indicating that the tonotopic map was not altered.

Lysosomal augmentation during aminoglycoside uptake in cochlear hair cells

Aminoglycoside antibiotics, such as kanamycin, have ototoxic side effects, which often result in degeneration of cochlear and vestibular hair cells in the inner ear. Cytotoxic effects of aminoglycosides, however, do not appear immediately after cellular uptake of aminoglycosides. In order to understand the mechanisms responsible for the delayed emergence of aminoglycoside ototoxicity, changes in lysosomal activities in cochlear hair cells were evaluated during a repeated administration of kanamycin by two methods. Electron microscopic localization of acid phosphatase (AcPase) revealed that AcPase started to accumulate in vesicles 27 h after the start of kanamycin administration. In addition, the number and size of AcPase-filled vesicles increased with repeated kanamycin doses. Confocal microscopic localization of the LysoTracker probe, a vital lysosomal marker, showed an increase in the size of lysosomes in hair cells that were treated with kanamycin. The temporal changes in the augmentation of lysosomes paralleled those in intracellular kanamycin levels. These results suggest that the intralysosomal compartments can accumulate extensive amounts of aminoglycosides, which might lead to lysosomal swelling and subsequent rupture.

Incomplete Recovery of Chicken Distortion Product Otoacoustic Emissions following Acoustic Overstimulation

Distortion product otoacoustic emissions (DPOAEs) were measured in chickens before and after exposure to a 525-Hz pure tone (120 dB SPL, 48 h). The exposure caused extensive hair cell loss and destroyed the tectorial membrane along the abneural edge of the basilar papilla in the low-to-mid-frequency region of the cochlea. Although the lesion was restricted, DPOAEs were greatly depressed at all frequencies immediately after the exposure. The high-frequency DPOAEs gradually recovered to preexposure values after the exposure; however, there was little or no improvement in DPOAEs at test frequencies equal to or slightly above the exposure frequency even after 16 weeks of recovery. By 28 days of recovery, the previously damaged region of the basilar papilla had been repopulated by hair cells and the lower honeycomb layer of the tectorial membrane had regenerated, but not the upper fibrous layer. The upper fibrous layer of the tectorial membrane was still missing after 16 weeks of recovery and the region of damage corresponded closely to the frequency regions where the DPOAEs were depressed.

Excitotoxic effect of kainic acid on chicken cochlear afferent neurons

The excitotoxic effects of kainic acid, a glutamate analog, on the auditory neurons in the chicken cochlea were assessed by light and transmission electron microscopy. Kainic acid was directly applied onto the round window of adult chickens and their cochleas were harvested 3 h after application. Transverse microscopic sections of the basilar papilla revealed swelling of afferent dendrites without any morphological changes in efferent endings. The regions of the basilar papilla damaged by kainic acid were localized in the apical 80% and primarily on the neural side where tall hair cells are located. The basal, abneural short hair cell region was devoid of damage. These results imply that glutamate is a primary neurotransmitter in chicken auditory afferent neurons that synapse on tall hair cells.

Radio frequency glow discharge and solid-phase lactoperoxidase-glucose oxidase beads as methods for etching ultra-thin plastic sections for immunoelectron microscopy

Etching techniques to prepare ultra-thin sections for immunoelectron microscopy have incorporated a variety of reagents to expose antigenic sites. In this paper involving 2 techniques for surface etching prior to immunoelectron microscopy, radio frequency glow discharge ( RFGD ) and solid-phase lactoperoxidase-glucose oxidase beads ( Enzymobeads ) are compared to conventional peroxide etching techniques. Measuring such parameters as intensity of granule disposition and titers of antibody resulting in detectable staining. RFGD and Enzymobeads were both superior to the conventional peroxide methodology. Non-specific absorption by ferritin under the conditions utilized was not a problem with Enzymobeads or RFGD method. In addition, RFGD may be useful in situations where peroxide susceptible antigens are under study.

Phenotypic variation of the antigen expression of the lipooligosaccharide of Neisseria gonorrhoeae

Gonococcal lipooligosaccharides (LOS) are a series of antigenically complex heteropolymers. To investigate whether all members of clonal populations of Neisseria gonorrhoeae express antigenically similar LOS, gonococcal strains 4505 and 220 were studied with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or co-migrating LOS unit(s) on SDS-Page. After sequentially staining organisms on formvar grids with these monoclonal antibodies and then staining with either 5nM or 15 nM colloidal gold spheres conjugated to goat antimurine IgM, 3 populations of cells could be identified among organisms derived from a single clone. Organisms which stained with 6B4 and 3F11 and organisms staining for either 6B4 alone or 3F11 alone. Immunofluorescent microscopy studies using rhodamine and fluorescein goat antimurine IgM conjugates to sequentially stain 3F11 and 6B4 also demonstrated these three populations. Fluorescent activated cell sorting studies of 3F11 coated strain 220 separated the population into two groups corresponding to organisms containing or lacking the 3F11 epitope. These studies indicate that clonally selected strains of N. gonorrhoeae contain members which have different phenotypic expression of different LOS antigens. Previously observed stability of the LOS antigenic and physicochemical structure suggest that this phenotypic variation is under environmental and/or genetic control.