蝙蝠耳蜗Corti器的电子显微镜观察

目的研究蝙蝠耳蜗基底膜毛细胞及静纤毛的特化现象。方法扫描电镜和透射电镜观察三种蝙蝠耳蜗毛细胞及静纤毛的表面亚显微结构形态。结果外毛细胞静纤毛特别的短，外毛细胞体呈瓶状或哑铃状，外毛细胞体被Deiters细胞的杯状膜包裹。结论我们观察到三种蝙蝠耳蜗外毛细胞体基本呈烧瓶状和哑铃状．明显有别于其他哺乳类耳蜗外毛细胞呈圆柱型的形态。这种毛细胞体的独特形态，可能是蝙蝠为适应高频回声定位的需要出现的特化现象。     

Spiral ganglion neuron survival and function in the deafened cochlea following chronic neurotrophic treatment

Cochlear implants electrically stimulate residual spiral ganglion neurons (SGNs) to provide auditory cues for the severe-profoundly deaf. However, SGNs gradually degenerate following cochlear hair cell loss, leaving fewer neurons available for stimulation. Providing an exogenous supply of neurotrophins (NTs) has been shown to prevent SGN degeneration, and when combined with chronic intracochlear electrical stimulation (ES) following a short period of deafness (5 days), may also promote the formation of new neurons. The present study assessed the histopathological response of guinea pig cochleae treated with NTs (brain-derived neurotrophic factor and neurotrophin-3) with and without ES over a four week period, initiated two weeks after deafening. Results were compared to both NT alone and artificial perilymph (AP) treated animals. AP/ES treated animals exhibited no evidence of SGN rescue compared with untreated deafened controls. In contrast, NT administration showed a significant SGN rescue effect in the lower and middle cochlear turns (two-way ANOVA, p < 0.05) compared with AP-treated control animals. ES in combination with NT did not enhance SGN survival compared with NT alone. SGN function was assessed by measuring electrically-evoked auditory brainstem response (EABR) thresholds. EABR thresholds following NT treatment were significantly lower than animals treated with AP (two-way ANOVA, p = 0.033). Finally, the potential for induced neurogenesis following the combined treatment was investigated using a marker of DNA synthesis. However, no evidence of neurogenesis was observed in the SGN population. The results indicate that chronic NT delivery to the cochlea may be beneficial to cochlear implant patients by increasing the number of viable SGNs and decreasing activation thresholds compared to chronic ES alone.     

Nitric oxide distribution and production in the guinea pig cochlea

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.     

Differentiation and distribution of acetylcholinesterase molecular forms in the mouse cochlea

The activities of the globular and asymmetric forms of acetylcholinesterase (AChE) were measured in the whole cochlea and cochlear turns of the developing postnatal mouse. The globular AChE forms (G4, G2 and G1) were present in each cochlear turn at birth. An asymmetric AChE form (A12) was detected in the midturn on the 4th postnatal day, and in the base and apex on the 7th postnatal day. The activities of all AChE molecular forms increased rapidly during the second postnatal week and reached a plateau by the 19th postnatal day. In the 26-day old mouse, G4 constitutes the largest proportion of total cochlear AChE (57%), G2/G1 being 37% and A12 being 6%. The distribution of the AChE forms among the different turns is as follows: the combined value of the activities of G2 and G1 AChE was the same in each turn; G4 was the major form in the base and midturn; and A12, the least abundant AChE form of all, was localized mainly in the base. Our results indicate that in the cochlea (1) the content of molecular forms is similar to that of other neuronal systems, (2) the expression of AChE molecular forms is developmentally regulated, and (3) the AChE isoenzymes develop and are distributed differentially along the cochlear length; resulting near maturity in the greater proportional expression of G4 and A12 in the base and midturn and G2/G1 in the apex.     

Muscarinic acetylcholine receptor binding in the guinea pig cochlea

Because of the possibility that olivocochlear bundles may use acetylcholine as an inhibitory transmitter, we examined the cochlea of the guinea pig for cholinergic receptor binding sites. Binding of the potent muscarinic antagonist [³H]quinuclidinyl benzilate (³H-QNB) showed that the cochlea has sites with the pharmacological specificity of muscarinic cholinergic receptors. Specific ³H-QNB binding was saturated at 0.8 nM and was half-saturated at 0.03 nM concentrations. Total ³H-QNB binding was reduced 70% in the presence of saturating doses of acetylcholine, oxotremorine and atropine, and half-maximal competition occurred at doses comparable to those at other muscarinic sites in the central and peripheral nervous systems. The nicotinic specific antagonist d-tubocurarine did not block ³H-QNB binding at concentrations known to be effective in electrophysiological experiments, indicating the sites measured here were not the mixed muscarinic-nicotinic receptor type postulated for some systems. Localization of binding sites, as done by hand microdissection methods, showed that basal cochlear turns had five times the number of sites as apical turns. Most of the sites were evenly divided between the bony modiolus and the auditory nerve, although some sites were measured in sensory tissue. These results support the idea that cholinergic communication occurs in the cochlea, but they are also consistent with the hypothesis that other receptor mechanisms may be involved in olivocochlear inhibition.     

Absence of mRNA encoding estrogen receptor in the rat cochlea

Based on changes in hearing thresholds and tinnitus that are co-related with the menstrual cycle, it has been suggested that the cochlea may respond directly to estrogen. For this to occur, the cochlea should express estrogen receptors. In situ mRNA hybridization was performed on normal female rat cochleas, using radiolabeled RNA probes complementary to mRNA encoding estrogen receptor, to determine whether estrogen receptors are present in the cochlea. Strong hybridization of the riboprobes to sections of uterus and hypothalamus indicated that the technique detected estrogen receptor mRNA. No hybridization to any cochlear tissues was observed. The results indicate that estrogen receptors are not expressed on cochlear cells, at least in rats. This in turn suggests that variation in cochlear responses during the estrus cycle are not the result of the direct effect of estrogen on the cochlea. Such variation may, however, be caused by systemic changes in fluid regulation induced by estrogen receptors at a distant site, or by other hormone receptors.     

Course and distribution of efferent fibers in the cochlea of the mouse.

The course, distribution and termination of single efferent fibers to the cochlea has been described in only a few animals and relatively few fibers have been studied with knowledge of their ipsilateral or contralateral origin. In order to examine the efferent fibers in the mouse, the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophoretically injected into one side of the brain stem near the location of known efferent nuclei. Examination of surface preparations of the cochlea revealed detailed information for both the lateral olivocochlear (LOC) and medial olivocochlear (MOC) systems. Many, but not all, fibers entered the cochlea within the intraganglionic spiral bundle (IGSB). The LOC fibers were restricted to the ipsilateral cochlea and rarely branched within the IGSB and osseous spiral lamina (OSL). In the organ of Corti, they traveled either basally or apically in the region of the inner hair cells (IHCs), spanning lengths up to 130 microns (basally) and 890 microns (apically). Terminal swellings of these fibers were ca 3.0 microns in diameter. Numerous en passant swellings were present where the fibers formed a plexus in the area of the IHCs. The MOC fibers followed a similar course in the IGSB and OSL, and within the OSL the fibers had few branches. Within the organ of Corti they traveled apically (up to 70 microns) in the nerve bundles located in the IHC area before they crossed the tunnel of Corti. In the region of the OHCs, 9% of the traceable fibers branched to innervate two to three OHCs while 91% appeared to innervate only one OHC. There was no discernible difference in the distribution of contralateral and ipsilateral MOC projections in terms of cochlear region or outer hair cell rows.     

P2X receptor immunoreactivity in the rat cochlea, vestibular ganglion and cochlear nucleus

P2X receptors have been reported to be involved in neurotransmission in both central and peripheral nerves. In the present study, polyclonal antibodies against P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6 were used to study the distribution of P2X receptors in rat cochlea and vestibulocochlear nerve pathways. It was found that in the vestibular ganglion all six types of antibodies stained the neurons to different intensities. The strongest signal was obtained with the P2X2 antibodies, followed by P2X3 antibodies. The other four antibodies produced weak signals, of approximately the same intensity. In the spiral ganglion, the six types of antibodies also stained almost all neurons. However, the rank order of intensity was different from that in the vestibular ganglion: the strongest signal was still obtained with P2X2 antibodies, followed by P2X4, P2X1, and P2X3 antibodies. The immunolabelling was much weaker with P2X5, and P2X6 antibodies compared with the other four types of antibodies. In the cochlea, besides the spiral ganglion neurons, other tissues such as stria vascularis, the organ of Corti and the tectorial membrane were labelled intensively with P2X2 antibodies only. High density P2X2 immunoreactivity was also observed in the vestibulocochlear nerve fibres. In the cochlear nucleus, neurons and nerve fibres were stained with the P2X2 antibodies, as were the neurons in the trapezoid body. These results suggested that P2 receptors, especially the P2X2 receptors, may play important roles in the signal transduction involved in the perception of sound and balance.     

Cellular and subcellular distribution of AMPA-type glutamate receptor subunits and metabotropic glutamate receptor 1alpha in the cochlear nucleus of the horseshoe bat (Rhinolophus rouxi)

Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) selective glutamate receptors (GluRs) are the main mediators of fast excitatory neurotransmission and composed of a variable combination of four different subunits (GluR1-4). The metabotropic glutamate receptor 1alpha (mGluR1alpha) is involved in plastic synaptic events. Since horseshoe bats strongly depend on temporal cues for acoustic imaging by echolocation and exhibit prominent species specific specializations of the cochlear nucleus (CN), the subunit distribution of AMPA selective GluRs and the distribution of mGluR1alpha was studied at the light and electron microscopic level with preembedding immunocytochemistry. Immunoreactivity to GluR1 was low throughout the CN. All types of projection neurons of the ventral CN expressed distinct GluR2/3 and GluR4 immunoreactivity with GluR4-labeling especially prominent in multipolar and octopus cell-like neurons of the posteroventral CN. The AMPA and metabotropic receptor inventory of the laminated ventral subdivision of the dorsal CN (DCNv) agreed with that reported in other mammals, whereas the specialized dorsal non-laminated subdivision of DCN (DCNd) lacked the prominent labeling for GluR2/3 and mGluR1alpha that characterizes cartwheel cells of DCNv. Distinct GluR2/3 and GluR4 immunoreactivity combined with low expression of mGluR1alpha immunoreactivity was characteristic for fusiform cells of DCNv and DCNd. Tuberculoventral cells of both the deep DCNv and the DCNd exhibited light to moderate GluR2/3 and GluR4 immunoreactivity. The staining patterns in DCNd thus indicate a loss of cerebellar-like microcircuits and a conservation of frequency specific circuitry of the deep and fusiform cell layers of the mammalian DCN.     

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.     

Na,K-ATPase expression in the mouse cochlea is not dependent on the mineralocorticoid receptor

This study was performed in order to test the hypothesis that the mineralocorticoid hormone stimulates the expression of Na,K-ATPase in the cochlea of the mouse. Immunohistochemistry was used to investigate the distribution of the mineralocorticoid receptor (MR) in the cochlea of the C57Bl/J6 mouse at different ages between gestational day 19 and postnatal day 30, and the occurrence and distribution of Na,K-ATPase in the inner ear of a mouse with a null mutation of the MR. Adult patterns of staining for MR were found as early as on gestational day 19 in the cochlea, with small changes thereafter. MR was detected in the same structures in the cochlea as Na,K-ATPase in earlier studies, where the amount of Na,K-ATPase increased after postnatal day 4. Thus there is latency between the increase of MR and the increase of Na,K-ATPase. In the cochlea of the MR deficient mouse, antibody labelling of Na,K-ATPase showed no significant difference as compared to the control wild type mouse. The hypothesis that mineralocorticoid hormone alone via MR stimulates the formation of Na,K-ATPase in the inner ear could not be confirmed by this study, and other regulating mechanisms must be considered.     

Topographical distribution of delay-tuned responses in the mustached bat inferior colliculus

In the mustached bat, delay-tuned neurons respond best to specific delays between the first harmonic frequency modulated (FM) component (FM1; 24-29 kHz) of the emitted biosonar pulse and a higher harmonic FM component in returning echoes (e.g. FM3, 72-89 kHz). These delay-tuned, combinatorial responses predominate in the inferior colliculus (IC) of the mustached bat. This study examined the topographical distribution of delay-tuned neurons in the 72-89 kHz frequency representation of the IC. We recorded and histologically localized 163 single units. Ninety units were facilitated and 41 were inhibited by the combination of two frequencies in the 24-29 kHz and 72-89 kHz ranges. The facilitatory responses were selective for delays up to 20 ms between the two signals. To determine if delay-tuned neurons were topographically organized, we plotted the dorsomedio-ventrolateral and caudo-rostral positions of each unit versus its best delay. Best delay was not correlated with either location. Response latency to best frequency tones was topographically organized, but was not correlated with best delay. This indicates that the latency axis in the IC is unrelated to the delay tuning of these combinatorial neurons. Because delay-tuned neurons are not topographically organized in the IC but are in the auditory cortex, our findings suggest that the creation and organization of delay-tuned neurons occur at different stages in the ascending auditory system.     

Immunoreactivity of endothelins and endothelin receptor in the stria vascularis of the mouse cochlea

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F1 +/+ mice and in that of W/Wv mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/Wv mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.     

Ryanodine receptor localisation in the mammalian cochlea: an ultrastructural study

Calcium-induced calcium release (CICR) in the mammalian cochlea has been suggested to enhance neurotransmitter release from inner hair cells and facilitate the efferent response in outer hair cells. Light microscopic evidence exists for the presence of ryanodine receptors in the organ of Corti but there is so far no information about their ultrastructural localisation. We have therefore used post-embedding immunogold labeling with antibodies that predominantly recognise ryanodine receptor isoforms 1 (RyR1) and 2 (RyR2) to investigate their distribution in rat cochleae. In inner hair cells, the highest levels of labeling were observed over an area of rough endoplasmic reticulum that lies in the cytoplasmic region beneath the nucleus; in outer hair cells, the cytoplasmic region above the nucleus displayed most labeling. Labeling was also associated with the subsurface cisternae adjacent to the lateral membranes of both types of hair cell, with the efferent terminals on the outer hair cells and was observed in adjacent supporting cells. Labeling in outer hair cells was significantly higher than that in inner hair cells or in the supporting cells. Our results support the presence of RyR1 in the cochlea but do not rule out the presence of other isoforms. CICR may be involved in the control of calcium levels in the base of the inner hair cells and supporting cells, and in the cholinergic efferent response and motile behaviour of the outer hair cells.     

Noise induces A1 adenosine receptor expression in the chinchilla cochlea

Adenosine plays a major cytoprotective role during ischemia and conditions of oxidative stress. Previous studies in our laboratory indicate that oxidative stress induces expression of the A1 adenosine receptor (A1AR) via activation of nuclear factor (NF)-kappaB. In this study, we tested whether noise exposure could induce oxidative stress and determine whether this induces expression of the A1AR in the chinchilla cochlea. Chinchillas were exposed to a 96 dB 4 kHz octave band of noise for 6 h of daily exposure, followed by an 18 h noise-free period. This noise paradigm resulted in threshold shifts of 10-60 dB over the frequency range (1-16 kHz) tested. Radioligand binding studies for the A1AR indicate a significant increase in receptor ( approximately 2-fold) expression soon after the first noise exposure period (usually within approximately 8 h of the initiation of noise), which gradually returned to basal levels by day 7. The rise in A1AR levels was followed by a significant increase in malondialdehyde levels by day 3, which also recovered by day 7. Assessment of the activity of NADPH oxidase in the cochlea indicates a significant increase in enzyme activity which was evident by approximately 8 h following initiation of noise exposure, and which persisted for at least up to day 3. Electrophoretic mobility shift assays indicate that the increase in A1AR was associated with a significant increase in NF-kappaB activity following noise exposure. We conclude that noise exposure induces A1AR expression, which might be mediated, in part, through generation of reactive oxygen species and activation of NF-kappaB.     

Immunocytochemical localization of AMPA selective glutamate receptor subunits in the rat cochlea

The localization of subunits of the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) selective glutamate receptor, termed Glutamate receptor (GluR) was examined in the rat cochlea using affinity purified polyclonal antibody to GluR subunits (GluR 1–4). GluR 2/3 and GluR 4 immunoreactive (IR) staining was observed in rat spiral ganglion cells, while GluR 1 IR was not. GluR 4 IR staining was also seen in puncta at inner and outer hair cell bases. These results suggest that GluR 2/3 and GluR 4 are components of excitatory amino acid synapses in the rat cochlea.