Immunofluorescence with Met-enkephalin and Leu-enkephalin antibodies in the guinea pig cochlea

We have applied an indirect immunofluorescence technique to cryostat sections of guinea pig cochleas using Met-enkephalin and Leu-enkephalin antisera. With both antisera, high immunofluorescence was seen in the inner spiral bundle, the tunnel spiral bundle and the intraganglionic spiral bundle. Immunoreactivity persisted to a dilution of 1/50 000. In three out of the twelve guinea pigs, immunoreactivity was observed under the outer hair cells, but only with the Met-enkephalin antiserum. Immunoreactivity of medium intensity was seen when the Met-enkephalin antiserum was pre-adsorbed by Leu-enkephalin. No immunoreactivity was seen when the Leu-enkephalin antiserum was pre-adsorbed by Met-enkephalin. After treatment of cochlear sections with acidic permanganate, immunoreactivity of medium intensity was still observed with the Leu-enkephalin antiserum. These observations indicate that Met-enkephalin and Leu-enkephalin co-exist in the cochlea in regions where efferent terminals are located, particularly in the inner spiral bundle below the inner hair cells.     

Proenkephalin and prodynorphin related neuropeptides in the cochlea

Dynorphin B (rimorphin), a proenkephalin B (prodynorphin)-derived peptide, and met-enkephalin-Arg6, Gly7, Leu8 (met-enkephalin octapeptide), a proenkephalin A-derived peptide, were identified in the mammalian cochlea by specific radioimmunoassays. The antisera are directed against unique sequences in the peptides, and this immunoreactivity cannot be ascribed to cross-reaction with any other known opioid peptides. Met-enkephalin octapeptide and dynorphin B can for this reason serve as reliable markers for the proenkephalin A- and proenkephalin B-derived peptides, respectively. Lesion studies in the cochlea indicate that dynorphin B is confined to olivocochlear efferents. It has not been determined if the dynorphin-containing neurons are the same as those known to contain enkephalin-related peptides, or if they may be cholinergic. Different, presumably inhibitory, neurotransmitters or modulators in the olivocochlear fibers create the possibility of separately modulating the effects of inner or outer hair cells on auditory nerve activity, and so becoming able to study their individual actions in audition. The olivocochlear fiber-hair cell-eighth nerve interaction may provide a valuable model for a complex multi-transmitter synaptic junction.     

Segregation of efferent projections to different turns of the guinea pig cochlea

Localized intracochlear injections of the fluorescent retrograde label diamidino yellow were used to investigate the organization of efferent projections from the brainstem to different turns of the cochlea, in the guinea pig. It was found that the location of small neurones within the lateral superior olive ipsilateral to the injection varied in a systematic manner when injections proceeded from base to apex of the cochlea. In addition, a cruder form of cochleotopic organization was present in that most of the large medial system efferent neurones were labelled only after injection into the 3 most basal turns of the cochlea. The decline of medial system efferent innervation proceeding from base to apex was most striking for the contralateral efferent neurones. The details of base to apex innervation density were different for the different nuclei of origin of the medial system, implying the existence of complex subsystems within the medial neurone population.     

Endogenous dynorphins: possible role in peripheral tinnitus

Tinnitus has been defined as the perceptual correlate of altered spontaneous neural activity occurring without an external auditory stimulus. Hyperacusis, defined as a collapse of tolerance to sound, is present in 40-86% of those who suffer from disabling forms of tinnitus. Both phenomena often are induced or exacerbated by physical or psychological stress. Biological systems known to regulate the body's overall response to stress use and release endogenous neuroactive opioid peptides. These stress-related neuromodulators consist of products derived from three genetically distinct precursor hormones. Two of these precursor hormones are proenkephalin and prodynorphin. Enkephalin and dynorphin-related peptides exist within the efferent olivocochlear systems (lateral and medial) of several mammalian species, including humans. Prodynorphin derivatives, however, may be restricted exclusively to lateral efferent neurons. Descending lateral efferent axons terminate solely on primary (type I) auditory dendrites innervating cochlear inner hair cells in most species. This action indicates that they play an important role in modulating auditory nerve sensitivity and spontaneous discharge. In a fashion similar to that exhibited by the observed excitatory mechanism of action of dynorphins in the spinal cord, sodium salicylate (aspirin) recently was shown to facilitate the excitatory effects of glutamate in the cochlea. This article provides support for a neurochemical model in which endogenous dynorphins may induce hyperacusis and can contribute to the induction, maintenance, or exacerbation of tinnitus in the auditory periphery by altering auditory type I neural excitability to glutamate.     

An ipsilateral cochlear efferent loop protects the cochlea during intense sound exposure

One suggested physiological function of the efferent nerve fibers innervating the cochlea is that they protect the cochlea against the effects of intense sound exposure. In order to test this hypothesis, we studied the effects of intense sound in the presence and in the absence of strychnine which blocks the efferent nerve fibers. The results show that in presence of strychnine an ipsilateral intense sound has a greater effect on the cochlea than in the absence of strychnine. We conclude that the ipsilateral cochlear efferents may act as protectors against intense sound exposure.     

γ─氨基丁酸在人体耳蜗的定位

应用免疫细胞化学方法，研究γ－氨基丁酸（ＧＡＢＡ）在人体耳蜗的定位。结果表明：ＧＡ－ＢＡ定位于人体耳蜗传出神经系统；是人体耳蜗传出神经递质。     

Computer-aided serial section reconstruction of nerve endings on the outer hair cells of the cochlea. Semithin sections vs. ultrathin sections

Afferent and efferent nerve endings on several outer hair cells in different turns and rows of the guinea pig cochlea were three-dimensionally reconstructed from serial semithin sections or ultrathin sections by means of a new computer system. In the basal turn, a single afferent ending was sometimes isolated from the others and surrounded by efferent endings, suggesting that this type of afferent ending may have properties different from the others. The advantages and disadvantages of semithin sections and ultrathin sections in computer-aided reconstruction were discussed and it was concluded that the semithin section technique will be useful in reconstructions of specimens from a wide range, whereas the ultrathin section technique will be better for reproducing fine structures such as the distribution of cell organelles as well as synaptic membrane specializations.     

卡铂导致毛细胞及其传出神经损害的耳蜗分析图

目的：介绍一种同时评估耳蜗传出神经和毛细胞的简便的组织化学技术。方法：首先应用脱氢酶染色选择性标记毛细胞,再用乙酰胆碱醌酶染色标记传出神经纤维,双重染色的耳蜗铺片样品在光学显微镜下沿着耳蜗基底膜的全长分别对毛细胞和穿越Ｃｏｒｔｉ隧道的传出神经纤维计数,根据卡铂耳中毒灰鼠的毛细胞及其传出神经纤维损伤的百分比制备耳蜗图。结果：耳蜗分析图充分显示;当绝大多数内毛细胞坏死以及部分外毛细胞坏死时,越隧道的传     

胆碱乙酰化酶在人体耳蜗的定位

应用免疫细胞化学方法，研究胆碱乙酰化酶在人体耳蜗的定位。结果发现，胆碱乙酰化酶定位于人体耳蜗传出神经系统。实验表明，乙酰胆碱是人体耳蜗的传出神经递质     

心钠素免疫反应物质在豚鼠耳蜗Corti器中的分布

目的 为了解豚鼠耳蜗Ｃｏｒｔｉ器组织中心钠素免疫反应（Ａｔｉａｌ ｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅｓ ｉｍｍｕｎｏｒｅａｃｔｉｏｎ，ＡＮＰ－ＩＲ）物质的分布及形态学特征。方法 对耳蜗切片组织采用免疫组织化学ＡＢＣ法进行研究。结果 Ｃｏｒｔｉ器中内侧第一、二排外毛细胞，Ｂｏｅｔｔｃｈｅｒ细胞和靠近螺旋韧带嵴的基底膜ＡＮＰ－ＩＲ为阴性，其余的组织细胞均为阳性反应。结论 ＡＮＰ在Ｃｏｒｔｉ器功能方     

Cochlear efferent neurons projecting to both ears in the chicken, Gallus domesticus.

Different retrograde neuroanatomical tracers were injected into each cochlea of adult chicken. The number of cells labeled in the cochlear efferent cell group found bilaterally within the caudal pontine reticular formation depended upon the tracer, with True Blue and Fluoro Gold yielding maximal average counts of 332 efferent neurons per injection. Double labeling of less than 1% of these cells was possible with the combination of True Blue and Diamidino Yellow. Thus the contribution of efferent neurons with axon collaterals projecting to both ears is not fundamentally different in birds and other vertebrates.     

Cochlear efferent neurones and protection against acoustic trauma: protection of outer hair cell receptor current and interanimal variability

We have measured the changes in neural and microphonic sensitivity in the basal turn of the guinea-pig cochlea produced by intense acoustic overstimulation (10 kHz, 115 dB SPL for 60 s and 150 s). As reported previously, the drop in neural and microphonic sensitivities observed after overstimulation were highly correlated [Patuzzi et al. (1989) Hear. Res. 39, 189-202]. Presentation of a non-traumatizing pure-tone to the contralateral ear (10 kHz, 80 dB SPL) during acoustic overstimulation reduced the amount of acoustic trauma measured using the neural response or the microphonic response. Transection of the medial olivo-cochlear system of efferent fibres at the floor of the fourth ventricle abolished this protective effect of contralateral sound and dramatically reduced the variability in the data. Since the low-frequency microphonic is a simple measure of the receptor current through the outer hair cells, and this current probably plays a part in enhancing the mechanical sensitivity of the cochlea, the protection of the microphonic we have observed suggests that the efferent system protects neural sensitivity by protecting the mechano-electrical transduction of outer hair cells. The drop in variability after sectioning the efferents also suggests that inter-animal variations in susceptibility to noise trauma may be a consequence of differing tonic activity of the efferents, and/or a variation in the sensitivity of the efferent pathway.     

Brainstem location of efferent neurones projecting to the guinea pig cochlea

Retrograde transport of horseradish peroxidase using tetramethylbenzidine as a chromagen was used to map the cell bodies of origin of the efferent projection to the cochlea in the guinea pig. Large numbers of small labelled neurones were found within the body of the lateral superior olivary nucleus (LSO) ipsi-lateral to the injected cochlea. Labelled cells within the contra-lateral LSO were extremely rare (1% or less). Large labelled neurones were found both ipsi-laterally and contra-laterally in the dorsomedial periolivary region, including the medial nucleus of trapezoid body and in the ventral and lateral nuclei of the trapezoid body and the ventral nucleus of lateral lemniscus. It was concluded that some aspects of the distribution of these efferent neurones in the guinea pig are similar to the situation in the rat, whilst others resemble more closely the arrangement found in cat.     

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.     

Reciprocal Synapses Between Outer Hair Cells and their Afferent Terminals: Evidence for a Local Neural Network in the Mammalian Cochlea

Cochlear outer hair cells (OHCs) serve both as sensory receptors and biological motors. Their sensory function is poorly understood because their afferent innervation, the type-II spiral ganglion cell, has small unmyelinated axons and constitutes only 5% of the cochlear nerve. Reciprocal synapses between OHCs and their type-II terminals, consisting of paired afferent and efferent specialization, have been described in the primate cochlea. Here, we use serial and semi-serial-section transmission electron microscopy to quantify the nature and number of synaptic interactions in the OHC area of adult cats. Reciprocal synapses were found in all OHC rows and all cochlear frequency regions. They were more common among third-row OHCs and in the apical half of the cochlea, where 86% of synapses were reciprocal. The relative frequency of reciprocal synapses was unchanged following surgical transection of the olivocochlear bundle in one cat, confirming that reciprocal synapses were not formed by efferent fibers. In the normal ear, axo-dendritic synapses between olivocochlear terminals and type-II terminals and/or dendrites were as common as synapses between olivocochlear terminals and OHCs, especially in the first row, where, on average, almost 30 such synapses were seen in the region under a single OHC. The results suggest that a complex local neuronal circuitry in the OHC area, formed by the dendrites of type-II neurons and modulated by the olivocochlear system, may be a fundamental property of the mammalian cochlea, rather than a curiosity of the primate ear. This network may mediate local feedback control of, and bidirectional communication among, OHCs throughout the cochlear spiral.     

Immunohistochemical localization of nerve growth factor receptor in the cochlea and in the brainstem of the perinatal rat.

Nerve growth factor receptor (NGF-R) localization was studied immunohistochemically in the cochlea and in the brainstem of the perinatal rat, using a specific monoclonal antibody directed against the rat NGF-R. In the cochlea, NGF-R immunoreactivity is positive during the whole perinatal period studied, and is located at the hair cell level, in fibers that reach the organ of Corti, in the intraganglionic spiral bundle and in some small bundles of fibers in the auditory nerve. In the brainstem, NGF-R is detected in auditory structures such as the ventral cochlear nucleus, the superior olivary complex, the nuclei of the trapezoid body and the trapezoid body. Many auditory structures labelled by the NGF-R antibody are implicated in the efferent cochlear innervation. These results suggest that NGF could be implicated in interactions between auditory receptors and efferent innervation of the developing cochlea. This coincides with findings on the immunohistochemical localization of NGF-like protein in the organ of Corti of the developing rat. Moreover, these observations could be related to an early prenatal development of auditory efferent innervation.     

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.     

Effect of Cortisporin Otic Suspension on Cochlear Function and Efferent Activity in the Guinea Pig

To predict cochlear receptor cell insult from therapeutic aminoglycoside antibiotics, we sought to determine whether the presence of subtoxic concentrations of Cortisporin otic suspension in the cochlea can be detected by measuring changes in efferent suppression of compound action potentials recorded from the round window. Olivocochlear efferent suppression in an earlier study was reversibly inhibited by injections of gentamicin in guinea pigs. Using that same technique, we found that 2.0 μL of Cortisporin otic suspension (neomycin, polymyxin B, hydrocortisone) placed on the round window of guinea pigs in varying concentrations causes a rapid, permanent alteration in thresholds. Solutions of Cortisporin otic suspension diluted to a concentration of 1:20 yield a reversible loss of efferent suppression without measurable changes in compound action potential. These data suggest that the presence of Cortisporin otic suspension can be detected functionally in the cochlea at very low concentrations prior to the development of a threshold shift.     

Untersuchungen zur efferenten Innervation der Cochlea beim Meerschweinchen mit Meerrettich-Peroxydase

Summary The origin of the efferent innervation of the cochlea was investigated by retrograde axonal transport of intracochlear administered horseradish peroxydase solution (HRP). Frontal frozen sections of the brain stem were reacted for the demonstration of HRP. Labeled neurons were found bilaterally in the superior olivary complex, especially in the ipsilateral lateral superior olivary nucleus (LSO) and in the ventral nucleus of the trapezoid body (VTB) contralaterally. Moreover, extraolivary origin of the efferent innervation of the cochlea could be demonstrated in the ventral nucleus of the lateral lemniscus (VLL) bilaterally.     

The effects of efferent activation on the acoustically and electrically evoked otoacoustic emission

The effects of efferent activation on the otoacoustic emission were measured in anesthetized guinea pigs. The otoacoustic emission (2F(1)-F(2)) was evoked by the conventional method of presenting either two continuous tones or a sinusoidal current to the round window (RW) of the cochlea. The efferent effects on the acoustically evoked emission are greatest at low stimulus levels and least for high levels. The efferent effects on the electrically evoked emission (EEOAE) are relatively constant across current levels. In each case, efferent activation resulted in an initial large reduction in the emission amplitude followed by a smaller and more constant reduction. Strychnine eliminated the efferent effects independent of the method of emission activation. Strychnine had no effect on the EEOAE, suggesting that the RW current did not evoke a local efferent effect. Slow versus fast efferent effects were observed in the recovery of the emission amplitude at the termination of efferent activation. Only a fast recovery in the emission amplitude was observed for stimuli below 10 kHz while the amplitude recovery had fast and slow components for stimuli presented above 10 kHz.