GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development.

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.

Excitotoxic effect of kainic acid on chicken otoacoustic emissions and cochlear potentials.

Kainic acid (KA) is a potent glutamate analog that can temporarily or permanently damage glutamatergic neurons. The purpose of the present study was to determine the short- and long-term effects of KA on chicken otoacoustic emissions and cochlear potentials. A chronic electrode was used to record the compound action potential (CAP), cochlear microphonic (CM), and the slow, positive neural potential (SPNP), a predominantly dc response. The CM, CAP, SPNP, and distortion product otoacoustic emissions (DPOAEs) were recorded before and after infusing 10 microl of a low dose (KA-L, 0.3 mM) or high dose (KA-H, 5 mM) of KA into scala tympani. KA caused a rapid and large reduction in CAP and SPNP amplitude in both the KA-H and KA-L groups; however, the CM and DPOAEs were largely unchanged. The amplitude of the CAP and SPNP in the KA-L group began to recover around 1 week post-KA, but was approximately 50% below normal at 4 weeks post-KA. In contrast, the CAP and SPNP showed no signs of recovery in the KA-H group. The results suggest that KA has no effect on the CM and DPOAEs generated by the hair cells, but selectively damages the CAP generated by the cochlear ganglion neurons. The reduction in the avian SPNP suggests that the response originates in the cochlear afferent neurons, unlike the summating potential (SP) in mammals that is generated in hair cells.

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.

Multiple actions of neurturin correlate with spatiotemporal patterns of Ret expression in developing chick cranial ganglion neurons.

The neurotrophic effects of neurturin (NRTN) on chick cranial ganglia were evaluated at various embryonic stages in vitro and related to its receptor expression. NRTN promoted the outgrowth and survival of ciliary ganglion neurons at early embryonic (E) stages (E6-E12), trigeminal ganglion neurons at midstages (E9-E16), and vestibular ganglion neurons at late stages (E12-E16). NRTN had no positive effects on cochlear ganglion neurons throughout development. In accordance with the time and order of onset in NRTN responsiveness, Ret protein was first detected in ciliary ganglia at E6, subsequently in trigeminal ganglia at E9, and in vestibular ganglia at E12. Ret was absent in E16 ciliary ganglia as well as in cochlear ganglia at all developmental stages that were tested. Exogenous application of retinoic acid induced NRTN responsiveness and Ret protein expression from E9 vestibular ganglion neurons, suggesting that retinoic acid can regulate Ret protein expression in peripheral sensory neurons in vitro. Ret was confined to the neuron cell body, whereas GFRalpha was localized predominantly in peripheral and central neurite processes. No noticeable change in GFRalpha expression was seen in any cranial ganglia throughout the developmental stages that were tested (E6-E16). These results demonstrate that NRTN exerts neurotrophic effects on different cranial ganglia at different developmental stages and that the onset and offset of NRTN responsiveness are regulated mainly by the spatiotemporal patterns of Ret, but not of GFRalpha receptors. The results also substantiate the recently emerging view that NRTN may be an essential target-derived neurotrophic factor for parasympathetic neurons during development.

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.

Hair cell regeneration and recovery of function in the avian auditory system.

Chickens were exposed to an intense pure tone that destroyed the hair cells and tectorial membrane in a crescent shaped patch along the abneural edge of the basilar papilla. During the following weeks, when the hair cells and tectorial membrane were regenerating, psychophysical and electrophysiological measures were obtained to assess the time course and degree of recovery. Immediately after the exposure, the behavioral thresholds were elevated 30-40 dB and auditory temporal integration was greatly reduced; however, both measures fully recovered by 28 days post-exposure. In addition, tone-on-tone masking patterns recovered to normal. Immediately after the exposure, the thresholds of single cochlear ganglion neurons were elevated more than 30 dB, tuning curves were broader than normal, two-tone rate suppression (TTRS) boundary slopes were shallower than normal and spontaneous activity was reduced. Threshold and spontaneous discharge rate fully recovered after the exposure. Tuning and TTRS also recovered significantly in most neurons; however, some units with characteristic frequencies (CFs) near the exposure frequency showed abnormal tuning and TTRS suppression. The regeneration of the hair cells and lower honeycomb layer of the tectorial membrane is associated with considerable recovery of function; however, the incomplete recovery of tuning and TTRS in some neurons may be linked to the incomplete regeneration of the tectorial membrane.

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.

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.

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.

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.

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 unit's 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.

Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide-digested P2 showed that two antibodies to the P2 protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains.

Characterization of pili expressed by Haemophilus ducreyi.

Twelve strains of Haemophilus ducreyi isolated primarily from chancroid outbreaks in North America were examined for the presence of pili by transmission electron microscopy. We identified piliated cells in 10 of the 12 strains. Pilin extracts were prepared from the mechanically sheared cells of the 12 H. ducreyi strains as well as the stably piliated H. influenzae strain R890 and its non-piliated parent R906. Pili were present in 12 out of 12 H. ducreyi extracts and in the R890 extract but not in the R906 preparation. Pili were purified by cycles of differential pH solubilization and crystallization. In SDS-PAGE, the preparation consisted predominantly of a protein whose apparent relative molecular mass was 24,000 (24 k), and an electron micrograph showed that the preparation contained pili. Three H. ducreyi strains were passed 52 times on agar plates, and extracts prepared from these strains contained pili. There was no evidence of binding of erythrocytes obtained from nine mammalian and avian species to colonies of one of the stably piliated H. ducreyi strains. We conclude that H. ducreyi expressed pili, that the relative molecular mass of the pilin monomer was 24 k, that pilus expression was not readily lost in passage and that H. ducreyi pili may not bind to an erythrocyte receptor.

Modification by sialic acid of Neisseria gonorrhoeae lipooligosaccharide epitope expression in human urethral exudates: an immunoelectron microscopic analysis.

Expression of lipooligosaccharide (LOS) antigenic determinants during human gonococcal infection was studied in secretions from seven men with gonococcal urethritis. Five monoclonal antibodies with distinct gonococcal LOS specificities and an H.8 lipoprotein monoclonal antibody were used in combination with immunogold electron microscopic analysis. The LOS epitope defined by antibody 6B7 was present on all seven strains in secretions and after in vitro growth. Gonococci from six of seven patients, when grown in vitro, expressed the 6B4 LOS epitope. The 6B4 epitope is a Gal beta 1-4-GlcNAc residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. This epitope was found unmodified on gonococcal LOS in urethral secretions from two patients. The unmodified epitope could not be demonstrated on organisms in five secretions. Neuraminidase digestion exposed the 6B4 epitope on organisms in these secretions and increased the 6B4 epitope density in the two secretions, which contained the unmodified epitope. These studies indicate that in vivo modification by sialylation of gonococcal LOS Gal beta 1-4-GlcNAc residue occurs during human infection.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide.

Gonococcal lipooligosaccharides (LOSs) are a series of antigenically complex heteropolymers. To investigate whether all members of clonally selected populations of Neisseria gonorrhoeae express antigenically similar LOS, we studied gonococcal strains 4505 and 220 with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or comigrating LOS unit(s) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent-antibody and immunoelectron microscopy studies indicated that all members of the clonally selected populations were not homogenous for the epitopes these antibodies recognized. Fluorescence-activated cell sorting studies of 3F11-coated strain 220 indicated that the density of epitope expression was a function of time of growth. The population could be separated into two broad groups corresponding to organisms staining strongly or weakly for the 3F11 epitope, and the epitope density decreased during the late-log and stationary phases of growth. Sequentially staining organisms on Formvar grids with 6B4 and 3F11, followed by staining with either 5- or 15-nm colloidal gold spheres conjugated to goat anti-mouse immunoglobulin M demonstrated the following populations of cells among organisms derived from a single clone: organisms which stained for both 6B4 and 3F11 epitopes and organisms which stained for either 6B4 epitopes alone or 3F11 epitopes alone. Immunofluorescence microscopy studies with rhodamine and fluorescein goat anti-mouse immunoglobulin M conjugates sequentially staining organisms on Formvar grids with 3F11 and 6B4 also demonstrated these three populations. Analysis of LOS preparations made over the last 5 years indicated no change in serotype antigen concentration or in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration pattern. These studies indicate that while clonally selected strains of Neisseria gonorrhoeae undergo phenotypic variation at the epitope level, the impact of this variation on the total LOS of the population has little overall effect on its antigenic or physicochemical properties.

Fimbriation of Haemophilus species isolated from the respiratory tract of adults.

Twenty-two clinical isolates of Haemophilus species were studied within two passages of their original isolation for the presence of fimbriae by negative-staining electron microscopy. Six isolates were identified as fimbriated, including three strains of nontypable Haemophilus influenzae, one strain of Haemophilus parainfluenzae, and two strains of Haemophilus haemolyticus. In fresh isolates of fimbriated strains of nontypable H. influenzae, approximately 40%-50% of cells had fimbriae; after five passages in vitro, less than 1% of the cells had fimbriae. Thus, a variety of Haemophilus species that colonize the human respiratory tract can be fimbriated, and this fimbriation is rapidly lost on passage in vitro.

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.