Cochlear neuroactive substances

We have reviewed the experiments done in our laboratory concerning various cochlear neuroactive substances. Data using chemical neuroanatomy and neurochemical techniques are described. They allow the identification and localization of neuroactive substances which could act as neurotransmitters and/or neuromodulators at the different types of synapses in the organ of Corti. Three hypotheses are presented: (1) the inner hair cells use glutamate as a neurotransmitter, but in addition to its excitatory properties, glutamate may also be involved in pathophysiological events affecting afferent auditory dendrites: (2) subpopulations of both the lateral and medial olivocochlear efferent systems can be differentiated by the neuroactive substances they may use: (3) the base and the apex of the cochlea can be distinguished on the basis of neurochemical data.     

Pharmacological alterations of the activity of afferent fibers innervating hair cells

To determine whether some of the substances that may be present in hair-cell sensory organs could affect neural activity in afferent fibers, we examined 56 compounds for the ability to alter the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis, the African clawed frog. These compounds included amino acids, glutamyl dipeptides, standard neurotransmitter candidates, and other constituents of tissues and body fluids. Substances found to be excitatory included some neutral amino acids (alanine, serine, threonine, asparagine, glutamine, and proline), ATP, carnosine, histidine, and barium chloride. Compounds that suppressed discharge included the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), serotonin, and gamma-glutamyl dipeptides. GABA and acidic amino acids (glutamate, aspartate, and cysteine sulfinate) produced a brief excitation followed by a suppression of discharge rate. Several of these substances were active at sufficiently low concentrations that their presence in body fluids may affect afferent fiber discharge rate under normal or pathological conditions.     

Cochlear neuroactive substances

Summary We have reviewed the experiments done in our laboratory concerning various cochlear neuroactive substances. Data using chemical neuroanatomy and neurochemical techniques are described. They allow the identification and localization of neuroactive substances which could act as neurotransmitters and/ or neuromodulators at the different types of synapses in the organ of Corti. Three hypotheses are presented: (1) the inner hair cells use glutamate as a neurotransmitter, but in addition to its excitatory properties, glutamate may also be involved in pathophysiological events affecting afferent auditory dendrites; (2) subpopulations of both the lateral and medial olivocochlear efferent systems can be differentiated by the neuroactive substances they may use; (3) the base and the apex of the cochlea can be distinguished on the basis of neurochemical data.Presented at the First European Congress of Oto-Rhino-Laryngology and Cervico-Facial Surgery, Paris, 26–29 September 1988     

Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig

The presence and localization of different neuropeptides and other putative neurotransmitters or -modulators were examined by immunohistochemistry in the cochleovestibular end organs and in neurons innervating them in rats and guinea pigs. In the organ of Corti neural elements beneath inner hair cells showed immunoreactivity for enkephalin (ENK), calcitonin gene-related peptide (CGRP), L-glutamate decarboxylase (GAD), substance P (SP) and tyrosine hydroxylase (TH). Nerve chalices of type I vestibular hair cells contained SP and GAD, but not consistently. SP was only occasionally observed in neuronal cell bodies of the 8th cranial nerve but fine fibers with different neuroactive substances were seen in the nerve trunk in the following relative numbers: TH greater than SP greater than CGRP greater than ENK. The present data demonstrate the presence of several different neuroactive substances in the rat and guinea pig inner ear suggesting a multiplicity of neurotransmitters or -modulators in this system.     

Differential modulation of spontaneous and evoked neurotransmitter release from hair cells: some novel hypotheses.

It has been generally accepted that even in the absence of mechanical stimulation of the transductional elements, a resting depolarizing current exists which is ultimately responsible for the spontaneous release of neurotransmitter. Movement of the transductional elements modulates this resting current and thereby the evoked release of neurotransmitter occurs. Recent data from our laboratory and others have led us to question whether the relationship between spontaneous and evoked neurotransmitter release is as simple as stated. Indeed, a variety of experimental manipulations appear to influence the two modes of release differently. Examination of our results and the results of others has led us to four hypotheses: 1. the two modes of neurotransmitter release are processed differently by the hair cells; 2. cyclic AMP is involved in spontaneous but not evoked neurotransmitter release; 3. there is a positive feedback step involving an excitatory amino acid and its receptor on the hair cell in evoked neurotransmitter release and; 4. different pools of calcium are involved according to the mode of release. Accordingly, there may be several biochemical steps between the transductional movement of the stereocilia at the apex of the hair cells and the ultimate release of the neurotransmitter at the base of these cells. Some of these biochemical steps are different depending on whether the mode of release is spontaneous or evoked. These biochemical steps may amplify or at least interact with the biophysical processes previously described in the hair cells.     

Bidirectional transduction in vertebrate hair cells: A mechanism for coupling mechanical and electrical processes

It has been proposed that the sharp frequency selectivity exhibited by cochlear hair cells and neurons of alligator lizards results from a mechanical resonance of the stereociliary-tectorial structures of hair cells. In contrast, in the red-eared turtle this selectivity has been attributed to an electrical resonance mechanism located in the hair-cell membrane. In this report a new mechanism is proposed, one which is consistent with observations in the lizard and turtle preparations and in which hair-cell resonances result from coupling of mechanical properties of hair-cell stereociliary-tectorial structures with electrical properties of the hair-cell membrane through a receptor membrane process that has bidirectional, mechanoelectric and electromechanical, transduction properties. This same mechanism could also be the physical basis for diverse phenomena observed in mammals, including changes in mechanical properties of the ear in response to electrical stimulation in the cochlea and central nervous system, and the presence of sustained, narrow-band, acoustic emissions in the ear canals of humans.     

The I' potential of the human auditory brainstem response to paired click stimuli

When stimulated with an appropriate stimulus, the hair cells of the organ of Corti depolarize, causing the release of a neurotransmitter substance, which excites afferent VIIIth nerve dendrites. It is reasonable to hypothesize that excitatory postsynaptic potentials (EPSPs) generated by the dendrites of the auditory nerve in turn initiate a compound action potential (CAP). The EPSP is thought to be the generator potential for the CAP, and may be recorded in auditory brainstem responses (ABRs) as the I' potential. Determining the anatomical origin of I' may enhance the sensitivity of the ABR technique in hair cell/dendrite/auditory nerve evaluations. Whether I' is of sensory or of neural origin is equivocal, and therefore I' is not well understood. To investigate this dilemma, ABRs were recorded from human subjects using standard and paired-click stimuli, and using subtraction methods to generate a derived ABR. Two early peaks, designated as I degree and I', occurred before wave I in the derived ABR. It was hypothesized that peaks I degrees and I' represent the summating potential and the generator potential, generated by the cochlea and VIIIth nerve dendrites, respectively.     

Flavin adenine dinucleotide is a major endogenous fluorophore in the inner ear

When illuminated with visible light, hair cells can exhibit autofluorescence (Lewis et al. [1982]Science 215, 1641–1643) concentrated in the basal pole near the synapses (Sento and Furukawa [1987] J. Comp. Neurol. 258, 352–367). The autofluorescence is enhanced by formaldehyde. The level of fluorescence is high enough to interfere with fluorescence microscopy of hair cells and to suggest that the fluorescent substance might have a particular role in hair-cell function. To identify this substance, we extracted a substance with formaldehyde-enhanced fluorescence from the inner ears of goldfish and purified it chromatographically. The substance copurified with FAD and had the same fluorescence emission spectrum. Two further results supported the identity of the endogenous fluorescent substance with FAD. First, as is the case with flavins, the autofluorescence in inner ear tissue examined within a few hours after fixation was reduced by addition of dithionite. Second, as is the case with the formaldehyde-enhanced fluorophore, the fluorescence of FAD was enhanced by formaldehyde. FAD accounted for 90% of flavins in goldfish inner ears; its concentration in the sensory epithelium was estimated to be about 30 nmol/g tissue weight, one of the highest tissue concentrations known. The FAD is probably associated with an unidentified flavoprotein concentrated in the basal, synaptic region of the hair cell.     

Initial changes in the sensory hair-cell membrane following aminoglycoside administration in a guinea pig model

This study demonstrates the initial changes affecting the sensory hair-cell plasma membranes in the vestibular end organs of gentamicin-treated guinea pigs by using a ruthenium red staining technique. First, 0.1 ml of a solution containing 5 mg gentamicin sulfate was injected into the middle ear. After 7 days, the sensory hair cell cilia were observed to be degenerating. The various stages of this degeneration process were classified into two types: the decrease in glycocalyx was designated type I fusion, while type II fusion was characterized by a bleb formation of the plasma membrane of the sensory hair cells, followed by a decrease in glycocalyx. The latter mechanism allowed plasma membrane contact, with subsequent fusion of the plasma membrane of neighboring sensory hair-cell cilia. The material also illustrates the degeneration of ciliary actin filaments. These findings suggest that the aminoglycoside affects both the glycocalyx and the plasma membrane, and that the decrease in glycocalyx may be the first sign of sensory hair-cell fusion.     

Serotonergic Innervation of the Organ of Corti

The olivocochlear efferent system of the mammalian cochlea, which is divided into two lateral and medial bundles, contains numerous neuroactive substances (acetylcholine, GABA, dopamine, enkephalins, dynorphins and CGRP). These have been located at the brainstem in neurons belonging to the lateral superior olive (lateral efferent system) or in neurons of the periolivary region around the medial superior olive and the trapezoid body (medial efferent system). All of these substances were found in well-characterized projections corresponding to lateral and medial nerve fibres and terminals which connect to the type I afferent dendrites and the outer hair cells, respectively. All could be involved in the modulation of the auditory process, as is suggested by the cochlear turnover increases observed in some of them (i.e. enkephalins or dopamine) induced by sound stimulation. Recently, the presence and distribution of serotonin-containing fibres has been included in the long list of cochlear neuroactive substances. However, its highly particular peripheral pattern of distribution together with the lack of response to sound stimulation could suggest that serotonergic fibres constitute a previously unknown cochlear innervation.     

Serotonergic innervation of the organ of Corti

The olivocochlear efferent system of the mammalian cochlea, which is divided into two lateral and medial bundles, contains numerous neuroactive substances (acetylcholine, GABA, dopamine, enkephalins, dynorphins and CGRP). These have been located at the brainstem in neurons belonging to the lateral superior olive (lateral efferent system) or in neurons of the periolivary region around the medial superior olive and the trapezoid body (medial efferent system). All of these substances were found in well-characterized projections corresponding to lateral and medial nerve fibres and terminals which connect to the type I afferent dendrites and the outer hair cells, respectively. All could be involved in the modulation of the auditory process, as is suggested by the cochlear turnover increases observed in some of them (i.e. enkephalins or dopamine) induced by sound stimulation. Recently, the presence and distribution of serotonin-containing fibres has been included in the long list of cochlear neuroactive substances. However, its highly particular peripheral pattern of distribution together with the lack of response to sound stimulation could suggest that serotonergic fibres constitute a previously unknown cochlear innervation.     

Initial changes in the sensory hair-cell membrane following aminoglycoside administration in a guinea pig model

Summary This study demonstrates the initial changes affecting the sensory hair-cell plasma membranes in the vestibular end organs of gentamicin-treated guinea pigs by using a ruthenium red staining technique. First, 0.1 ml of a solution containing 5 mg gentamicin sulfate was injected into the middle ear. After 7 days, the sensory hair cell cilia were observed to be degenerating. The various stages of this degeneration process were classified into two types: the decrease in glycocalyx was designated type I fusion, while type II fusion was characterized by a bleb formation of the plasma membrane of the sensory hair cells, followed by a decrease in glycocalyx. The latter mechanism allowed plasma membrane contact, with subsequent fusion of the plasma membrane of neighboring sensory hair-cell cilia. The material also illustrates the degeneration of ciliary actin filaments. These findings suggest that the aminoglycoside affects both the glycocalyx and the plasma membrane, and that the decrease in glycocalyx may be the first sign of sensory hair-cell fusion.     

Substance P in the acoustic area of the cortex during neuronal development and maturation

Substance P has been proved an important neurotransmitter and neuromodulator, serving not only as an excitatory transmitter of the first afferent synapse of pain pathways but as a valuable neuromodulator in the primary cortical areas of sensosensorial integration. In the human acoustic cortex, substance P has been found in the presynaptic terminals as well as in the cell bodies of numerous polyhedral, triangular and bipolar neurons. Using histochemical techniques, we found recently that substance P is first accumulated in the neuronal elements of the human acoustic cortex in 18-gestational-week (GW) fetuses. Most of substance P is distributed in the neurons of the 3rd and 4th cortical layers, mainly in the cell bodies, the initial part of the axons and the axonic terminals. In 24-GW fetuses, substance P is found in the 2nd cortical area inside the cell body of the granular and bipolar neurons. In 28-GW fetuses, substance P is found in the pre- and postsynaptic terminals. We would suggest that substance P is accumulated increasingly as a function of the neuronal maturity of the primary acoustic cortical area, serving mainly as a neuromodulator.     

Stiffness of sensory-cell hair bundles in the isolated guinea pig cochlea

Stiffness of hair bundles on cochlear hair cells was measured in turns 2, 3 and 4 of isolated preparations of the guinea-pig organ of Corti maintained in tissue culture medium. Defined as the force required to produce a linear 1.0 micron deflection of the hair-bundle tip, stiffness is greater for deflection in the excitatory than in the inhibitory direction. The excitatory-to-inhibitory ratio for inner hair cells (IHC) is significantly lower than the ratio for outer hair cells (OHC). Hair-bundle stiffness decreases radially from the first to third rows of OHC. Over the measurement range of 9.0-18.0 mm from the stapes hair-bundle stiffness decreases much more for OHC (88-97%) than for IHC (50%). Although an increase in hair-bundle length with distance from the stapes accounts for some of the observed stiffness decrease, the major decrease is due to an increase in compliance of the sensory-hair attachment to the hair-cell surface.     

The efferent modulation of mammalian inner hair cell afferents.

The results of immunocytochemical, enzymatic and electrophysiological studies have indicated that acetylcholine and GABA may act as neurotransmitters in lateral olivocochlear efferent endings on inner hair cell afferent dendrites. Since spike activity can be recorded in the dendritic region of inner hair cells, microiontophoretic techniques were used testing the possible neurotransmitter candidates, acetylcholine and GABA, on spontaneous and induced firing of the afferent dendrites. The experiments were carried out in anaesthetised guinea-pigs, the third and fourth turns of the cochlea being exposed for electrode penetration. Ejection of acetylcholine resulted in a pronounced dose-dependent increase in subsynaptic spiking activity. Furthermore, acetylcholine enhanced glutamate-induced activity. In contrast, even at high doses, GABA had very little effect on the spontaneous cochlear firing rate. When the firing rate had first been enhanced by glutamate or N-methyl-D-aspartate, however, this activation could be reduced by the ejection of GABA. A similar reduction was observed when the firing rate had been enhanced with acetylcholine. The results of our studies support the hypothesis that these substances are involved in efferent neurotransmission on inner hair cell afferent fibres. It should be pointed out, however, that besides acetylcholine and GABA, several opioids such as enkephalins and dynorphins seem to be involved in efferent cochlear innervation.     

Die Rolle der kochleären Neurotransmitter in Bezug auf Tinnitus

Pathologic changes in the cochlear neurotransmission, e.g. as a result of intensive noise exposure or ototoxic drugs, can be a factor in the development of tinnitus. The efficiency of inhibitory and excitatory neurotransmitters may then be modulated at the switching points. Glutamate is the most important afferent neurotransmitter within the inner ear. A massive glutamate release induced by cochlear damage may result in excitotoxicity and irrevocable cell death. Efferent cochlear neurotransmitters include dopamine, gamma aminobutyric acid (GABA), acetylcholine (ACH) and serotonin. Dopamine and GABA are inhibitory transmitters that may protect the cochlea from excitotoxicity. ACH, like GABA, reduces the stiffness of the outer hair cells and increases their motility. Serotonin is a neuromodulator of the cholinergic and GABAergic innervation within the cochlea and can inhibit glutamatergic impulses. Our understanding of neurotransmission in the cochlea has been extended by advances in molecular biology, which has given rise to new approaches in the treatment of tinnitus. As there are several types of tinnitus, differing in aetiology and development, our present challenge is to achieve precise identification of the cause in individual cases of tinnitus.     

Age-related changes in cochleas of mongolian gerbils

The effects of aging on the gerbil cochlea were studied in 16 animals raised in a quiet environment. Animals were tested at ages ranging from 33 to 36 months, the approximate average lifespan of gerbils in our colony. Hearing sensitivity was assessed by measures of whole-nerve compound action potential (CAP) thresholds and surface preparations of the organ of Corti were subsequently examined by light microscopy for losses of sensory hair cells. These quiet-aged animals showed a wide range of hair-cell losses and threshold shifts. Outer hair cells often showed significant losses while inner hair cells were rarely absent. All animals had some threshold shift, especially at frequencies above 4 kHz. These shifts ranged from 1 to 68 dB. At high frequencies, threshold shifts often occurred without hair-cell losses at corresponding cochlear locations. At low frequencies, threshold shifts seldom reflected the losses of hair cells commonly found in the cochlear apex. Thus, the correlation of specific hair-cell losses and CAP threshold shifts at corresponding frequencies was poor. On the other hand, the total number of missing hair cells, irrespective of location, was a good, general indicator of the hearing capacity in a given ear. It appears that the factor or factors that makes cochleas susceptible to hair-cell loss with increasing age also affects other cochlear mechanisms that are necessary for normal functioning of the ear.     

Distribution of efferent cholinergic terminals and α-bungarotoxin binding to putative nicotinic acetylcholine receptors in the human vestibular end-organs

Although acetylcholine (ACh) has been identified as the primary neurotransmitter of the efferent vestibular system in most animals studied, no direct evidence exists that ACh is the efferent neurotransmitter of the human vestibular system. Choline acetyltransferase immunohistochemistry (ChATi), acetylcholinesterase (AChE) histochemistry, and α-bungarotoxin binding were used in human vestibular end-organs to address this question. ChATi and AChE activity was found in numerous bouton-type terminals contacting the basal area of type II vestibular hair cells and the afferent chalices surrounding type I hair cells; α-bungarotoxin binding suggested the presence of nicotinic acetylcholine receptors on type II vestibular hair cells and on the afferent chalices surrounding type I hair cells. This study provides evidence that the human efferent vestibular axons and terminals are cholinergic and that the receptors receiving this innervation may be nicotinic. Laryngoscope, 105:1167-1172, 1995     

A biochemical model of peripheral tinnitus

Subjective tinnitus may be defined as the perceptual correlate of altered spontaneous neural activity occurring in the absence of an externally evoking auditory stimulus. Tinnitus can be caused or exacerbated by one or more of five forms of stress. We propose and provide evidence supporting a model that explains, but is not limited to, peripheral (cochlear) tinnitus. In this model, naturally occurring opioid dynorphins are released from lateral efferent axons into the synaptic region beneath the cochlear inner hair cells during stressful episodes. In the presence of dynorphins, the excitatory neurotransmitter glutamate, released by inner hair cells in response to stimuli or (spontaneously) in silence, is enhanced at cochlear N-methyl-D-aspartate (NMDA) receptors. This results in altered neural excitability and/or an altered discharge spectrum in (modiolar-oriented) type I neurons normally characterized by low rates of spontaneous discharge and relatively poor thresholds. It is also possible that chronic exposure to dynorphins leads to auditory neural excitotoxicity via the same receptor mechanism. Finally, the proposed excitatory interactions of dynorphins and glutamate at NMDA receptors need not be restricted to the auditory periphery.     

Mechanisms of rapid sensory hair-cell death following co-administration of gentamicin and ethacrynic acid

Concurrent administration of a high dose of gentamicin (GM; 125 mg/kg IM) and ethacrynic acid (EA; 40 mg/kg IV) results in rapid destruction of virtually all cochlear hair cells; however, the cell death signaling pathways underlying this rapid form of hair-cell degeneration are unclear. To elucidate the mechanisms underlying GM/EA-mediated cell death, several key cell death markers were assessed in the chinchilla cochlea during the early stages of degeneration. In the middle and basal turns of the cochlea, massive hair-cell loss including destruction of the stereocilia and cuticular plate occurred 12 h after GM/EA treatment. Condensation and fragmentation of outer hair-cell nuclei, morphological features of apoptosis, were first observed 5–6 h post-treatment in the basal turn of the cochlea. Metabolic function, reflected by succinate dehydrogenase histochemistry and mitochondrial staining, decreased significantly in the basal turn 4 h following GM/EA treatment; these early changes were accompanied by the release of cytochrome c from the mitochondria into the cytosol and intense expression of initiator caspase-9 and effector caspase-3. GM/EA failed to induce expression of extrinsic initiator caspase-8. These results suggest that the rapid loss of hair cells following GM/EA treatment involves cell death pathways mediated by mitochondrial dysfunction leading to the release of cytochrome c, activation of initiator caspase-9 and effector caspase-3.