Muscarinic receptors modulate intracellular Ca(2+) concentration in hyaline cells of the chicken basilar papilla.

Nonsensory hyaline cells border the sensory epithelium of the auditory end-organ (basilar papilla) in birds and reptiles. Their innervation by cochlear cholinergic efferent fibers and the presence of contractile proteins suggest that hyaline cells may actively regulate basilar membrane mechanics. The cholinergic pharmacology of hyaline cells was studied by measuring the intracellular calcium concentration ([Ca(2+)](i)) of fura-2-loaded cells in the chicken cochlea in vitro. Superfusion of the cholinergic agonist carbachol produced a dose-dependent increase in hyaline cell [Ca(2+)](i) (EC(50)=1.05 micromol l(-1)) and small responses in short hair cells. Calcium increases in hyaline cells were evoked by the muscarinic agonists oxotremorine (10 micromol l(-1)) and muscarine (100 micromol l(-1)) whereas nicotine (100 micromol l(-1), 200 micromol l(-1)) was without effect. Carbachol-evoked responses were blocked by the muscarinic antagonist atropine (>or=10(-13) mol l(-1)) and were unaffected by the nicotinic antagonists d-tubocurare (100 micromol l(-1), 1 mmol l(-1)) and hexamethonium (100 micromol l(-1)). Responses persisted in the absence of extracellular Ca(2+) and were abolished by thapsigargin (1 micromol l(-1)). These results indicate that the cholinergic-stimulated increase in hyaline cell [Ca(2+)](i) is due to a muscarinic-mediated release of Ca(2+) from intracellular stores. This is the first evidence that hyaline cells possess a muscarinic receptor whose activation causes mobilization of intracellular Ca(2+).

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons.

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca2+ indicator dye Fura-2 was used to measure Ca2+ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca2+ responses were evoked by kainic acid (KA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D-aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca2+]i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca2+ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca2+]i were carried by Ca2(+)-permeable receptor channels. Significantly smaller changes in [Ca2+]i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca2+ free-EGTA--buffered media, including L-cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclo-pentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca2+]i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca2+]i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca2+ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca2+]i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca2+]i to increase in NM neurons. Presumably, each route represents a means by which Ca2+ can alter cellular processes.

Activity-dependent regulation of [Ca2+]i in avian cochlear nucleus neurons: roles of protein kinases A and C and relation to cell death.

Neurons of the cochlear nucleus, nucleus magnocellularis (NM), of young chicks require excitatory afferent input from the eighth nerve for maintenance and survival. One of the earliest changes seen in NM neurons after deafferentation is an increase in intracellular calcium concentration ([Ca2+]i). This increase in [Ca2+]i is due to loss of activation of metabotropic glutamate receptors (mGluR) that activate second-messenger cascades involved in [Ca2+]i regulation. Because mGluRs are known to act via the phospholipase C and adenylate cyclase signal transduction pathways, the goal of this study was to determine the roles of protein kinases A (PKA) and C (PKC) activities in the regulation of NM neuron [Ca2+]i by eighth nerve stimulation. Additionally, we sought to determine the relationship between increased [Ca2+]i and cell death as measured by propidium iodide incorporation. [Ca2+]i of individual NM neurons in brain stem slices was monitored using fura-2 ratiometric fluorescence imaging. NM field potentials were monitored in experiments in which the eighth nerve was stimulated. Five hertz orthodromic stimulation maintained NM neuron [Ca2+]i at approximately 110 nM for 180 min. In the absence of stimulation, NM neuron [Ca2+]i increased steadily to a mean of 265 nM by 120 min. This increase was attenuated by superfusion of PKC activators phorbol-12,13-myristate acetate (100 nM) or dioctanoylglycerol (50 microM) and by activators of PKA: 1 mM 8-bromoadenosine-3',5'-cyclophosphate sodium (8-Br-cAMP), 50 microM forskolin or 100 microM Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine. Inhibition of PKA (100 microM Rp-cAMPS) or PKC (50 nM bisindolymaleimide or 10 microM U73122) during continuous orthodromic stimulation resulted in an increase in NM neuron [Ca2+]i that exceeded 170 and 180 nM, respectively, by 120 min. Nonspecific kinase inhibition with 1 microM staurosporine during stimulation resulted in an [Ca2+]i increase that was greater in magnitude than that seen with either PKA or PKC inhibition alone, equal to that seen in the absence of stimulation, but much smaller than that seen with inhibition of mGluRs. In addition, manipulations that resulted in a [Ca2+]i increase >/=250 nM resulted in an increase in number and percentage of propidium iodide-labeled NM neurons. These results suggest that eighth nerve activity maintains [Ca2+]i of NM neurons at physiological levels in part via mGluR-mediated activation of PKA and PKC and that increases in [Ca2+]i due to activity deprivation or interruption of the PKA and PKC [Ca2+]i regulatory mechanisms are predictive of subsequent cell death.

Relationship between frequency of spontaneous bursting and tonotopic position in the developing avian auditory system.

Neural activity in the developing brainstem auditory pathway of the chick embryo is dominated by a rhythmic pattern of spontaneous discharge. Neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL), second and third order auditory nuclei, discharge spontaneously in synchronous bursts at periodic intervals. Rhythmic bursting is present as early as embryonic day 14 (E14), shortly after the onset of functional synaptogenesis, and gives way to an adult-like, steady level of firing on E19, two days prior to hatching. In the present experiment, multiple-unit recording techniques were used in E17 and E18 embryos to examine the relationship between rate of rhythmic bursting and tonotopic position in NM and NL. The mean rate of rhythmic bursting ranged from 0.21-0.71 Hz. Bursting rate varied systematically as a function of position, being faster at progressively higher frequency regions of the nuclei at both E17 (r = 0.75) and E18 (r = 0.86). In addition, the rate of bursting at a given location in the nuclei increased during development. The presence of a systematic relationship between the rate of rhythmic bursting and tonotopic location suggests that the spatio-temporal pattern of spontaneous discharges could provide developmental cues for the spatial ordering of auditory projections.

Deafferentation increases the intracellular calcium of cochlear nucleus neurons in the embryonic chick.

1. Ratiometric fura-2 imaging was used to measure the intracellular calcium concentration ([Ca2+]i) of neurons in the embryonic avian cochlear nucleus, nucleus magnocellularis (NM), after an in ovo unilateral cochlea removal (deafferentation). 2. The mean [Ca2+]i of NM neurons receiving normal input was 113 nM. 3. Deafferentation increased the mean [Ca2+]i of NM neurons to 247, 311, 339, and 314 nM at 1, 3, 6, and 12 h after cochlear removal, respectively. These values did not differ significantly. 4. The percent frequency distribution of deafferented NM neuron [Ca2+]i shifts away from normative levels toward higher [Ca2+]i at 1 and 3 h after cochlear removal, but shifts back toward normative levels at 6 and 12 h after cochlear removal. 5. This increased [Ca2+]i following cochlear removal temporally coincides with well-characterized changes in NM neurons following activity deprivation. 6. These data suggest that deregulation of [Ca2+]i homeostasis plays a key role in NM neuron degeneration and death following activity deprivation.

Effects of middle ear pressure on frequency representation in the central auditory system.

Changes in middle ear pressure (MEP) are known to produce an attenuation of sound transmission through the outer and middle ear, but the effects on frequency representation in the auditory system have not previously been studied. This issue is of particular interest because of changes in MEP occurring during episodes of otitis media. We have investigated the effect of changes in MEP on the tuning of neurons in the inferior colliculus (IC) of the gerbil to calibrated tone stimulation of the contralateral, pressurized ear. Both negative and positive non-atmospheric MEP produced an elevation of neural thresholds that was inversely related to IC neuron best frequency (BF). A robust, linear relationship was found between BF at atmospheric MEP (control) and BF at -20 daPa MEP. Higher resolution analysis was performed on a sub-sample of neurons that had particularly stable BFs with repeated, control MEP. For the majority of these neurons, alternation of MEP between control and -20 daPa had no effect on BF. However, a few neurons showed small (up to 5%), significant shifts in BF with -20 daPa MEP. These results are consistent with previous reports of the effects of MEP on spontaneous otoacoustic emissions. We conclude that non-atmospheric MEP acts as a high-pass filter on the input to the cochlea, but does not change the frequency organization of the auditory system to any marked extent.

Cochlear microphonic measurements of interaural time differences in the chick.

The major cues for the sound localization are the interaural differences in the timing and intensity of acoustic information. This poses a difficult coding problem for animals with relatively small heads, such as birds, because interaural time differences (ITDs) would have a small range and magnitude and interaural intensity differences (IIDs) would be significant for only high frequency sounds. It has been suggested that this coding problem is mitigated in birds by an enhancement of ITDs and IIDs resulting from the acoustic coupling of the two middle ear cavities through an interaural canal. In this report, the functional ITDs for sounds at different azimuthal locations were recorded in young chicks, and the contribution of middle ear acoustic coupling was evaluated. ITDs were calculated from simultaneous cochlear microphonic (CM) recordings evoked by pure tone stimuli. These effective ITDs were larger than predicted by the physical separation of the two ears, and this enhancement was more pronounced at low (0.8 and 1 kHz) than at high (2 and 4 kHz) frequencies, reaching maximum values of approximately 180 and 100 microseconds, respectively. The amplitude of the CM also varied as a function of sound source location. This variation was as much as +/- 30%, even for the low frequency tones. This suggests that IID cues are also available to the chick. To determine the contribution of middle ear acoustic coupling to the timing and amplitude of the CM response, the CM in one ear was measured prior to, and following occlusion of the contralateral external auditory canal. The cochlear microphonic from the ear distal to the sound source advanced in time and increased in amplitude when the ear proximal to the sound source was sealed. These effects were more pronounced for low frequency sounds. These findings confirm that acoustic coupling of the middle ear cavities plays a role in enhancing sound localization cues in the chick.

Rhythmic spontaneous activity in the developing avian auditory system.

Microelectrode recordings of spontaneous multiple unit activity were made from nucleus magnocellularis (NM) and nucleus laminaris (NL), second- and third-order nuclei in the chick auditory system, between 14 and 19 d of incubation (E14-E19). Spontaneous firing in E14-E18 embryos occurred in synchronous bursts at periodic intervals. A rhythmic pattern of spontaneous firing was also observed in the auditory nerve but not in nonauditory regions of the brain-stem. The mean interburst interval in NM and NL decreased from 4.9 sec at E14-E15 to 2.1 sec at E18. By E19, 2 d prior to hatching, synchronous bursting was replaced by an unpatterned, steady level of firing comparable to the background discharge that is present in NM and NL of hatchling birds. Bursting was not correlated with heart beat or respiration and was not affected by removal of the middle-ear ossicle. Rhythmic bursting could be reset, blocked, or induced by sound stimulation. Cochlea removal or pharmacological blockade of auditory nerve activity with TTX eliminated bursting. These results indicate that the synchrony and rhythmicity of impulse firing reflect normal physiological activity, most likely of cochlear origin. The present findings show that spontaneous activity in the embryonic avian auditory system, like that in the immature mammalian visual pathway (Maffei and Galli-Resta, 1990; Meister et al., 1991), occurs in a synchronously rhythmic pattern. This similarity raises the possibility that such activity may be a general feature of early sensory system development. Patterned spontaneous firing in the chick takes place during a period of embryogenesis when auditory thresholds are high and when it is unlikely that physiological function in ovo is influenced significantly by normally occurring levels of airborne sound. Brainstem auditory neurons undergo substantial changes in structure and innervation during this same period. It is speculated that the temporal pattern of spontaneous discharge may provide cues that contribute to these developmental events.

Aberrant projection induced by otocyst removal maintains normal tonotopic organization in the chick cochlear nucleus.

Nucleus magnocellularis (NM), a second-order nucleus in the chick auditory system, is topographically and tonotopically organized. The basilar papilla (cochlea) projects onto the ipsilateral NM via the auditory nerve. The anteromedial region of NM is innervated by the proximal end of the basilar papilla and responds to high-frequency sounds; more posterolateral regions receive input from more distal locations along the papilla and respond to progressively lower frequencies. NM projects exclusively to the third-order neurons of nucleus laminaris (NL). Otocyst removal prevents the formation of the ipsilateral cochlea and cochlear nerve and results in the development of an aberrant functional projection from the contralateral NM to the "deafferented" NM on the operated side of the brain (Jackson and Parks, 1988). In the present experiment, the otocyst was removed unilaterally and the tonotopic organization of the deafferented NM was physiologically mapped at 17-18 d of embryonic age (E17-E18). Quantitative analyses revealed that the frequency organization of the deafferented NM is almost identical to that in normal embryos. Progressively higher characteristic frequencies were recorded at successively more anterior and more medial locations in the nucleus, and the orientation of the tonotopic axis was indistinguishable from normal. Furthermore, the correlation between characteristic frequency and anatomical location is comparable in the deafferented (r = 0.91) and normal (r = 0.87) NM. The only noticeable discrepancy is that characteristic frequencies in NM on both sides of the brain of operated embryos are higher than the frequencies observed previously at comparable regions of the nucleus in unoperated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of muscarinic cholinergic receptors stimulates inositol phosphates synthesis in the developing avian cochlear duct.

We previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCl, the accumulation of (3H)-IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post-hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady-state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between E15 and E19, by a transient increase in IPs synthesis. This rise peaks at E16 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose-dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity.

Hair cell loss in the avian cochlea partially recovers following both acoustic trauma and aminoglycoside intoxication. DNA labeling with tritiated thymidine has shown that the restoration of cell number following acoustic trauma results from the production of new hair cells by mitotic division. The purpose of the present study was to determine if mitosis also contributes to the recovery of hair cell number which occurs following aminoglycoside intoxication. Chickens received daily injections of either gentamicin sulfate or distilled water for 10 consecutive days. During the latter 7 days of this period, all birds were also injected with [3H]thymidine. Following postinjection survival periods of 3 or 6 days, one papilla from each bird was processed for autoradiography and the other for scanning electron microscopy (SEM). Incorporation of [3H]thymidine was seen over hair cells and support cells in experimental papillae in regions of hair cell loss. No labeling was seen outside of damaged regions or in the papillae of control birds. SEM showed that damaged regions in experimental birds contained cells similar in appearance to developing auditory hair cells in avian embryos. These results show that the restoration of hair cell number following aminoglycoside toxicity results from the production of new cells by mitosis.

Reduction and recovery of neuronal size in the cochlear nucleus of the chicken following aminoglycoside intoxication.

The effect of aminoglycoside intoxication on the cross-sectional area of neurons in nucleus magnocellularis (NM) was studied in neonatal chickens. Birds received daily injections of 100 mg/kg body weight of gentamicin for 10 consecutive days. Cell area was measured at five different tonotopic regions along the posterior-to-anterior dimension of NM (low-to-high frequency) after post-treatment survival times of 8, 23 and 40 days. Gentamicin caused a reversible reduction of cell area that varied as a function of location and survival time. Significant decreases of cell area occurred only in the rostral half of the nucleus. Cell area was reduced at 8 and 23 days survival and recovered to near control values by 40 days post-treatment. Body weight, brain weight and the cross-sectional area of cerebellar Purkinje neurons were also reduced but did not recover. The present results show that aminoglycoside toxicity can affect auditory neurons in the brain. It is suggested that two factors contributed to the changes in NM neuron size: (1) Processes specifically related to the loss and regeneration of cochlear hair cells, most likely changes in afferent activity. (2) A general retardation in growth.

Shift of tonotopic organization in brain stem auditory nuclei of the chicken during late embryonic development.

The tonotopic organization of nucleus magnocellularis and nucleus laminaris, second and third order nuclei in the avian auditory system, was mapped in 19-20-day old chick embryos (E19-20). The characteristic frequency recorded at any given location in each nucleus was intermediate between the frequencies observed previously at E17 and one day after hatching. This indicates that tonotopic organization changes during the embryonic as well as the postnatal development of hearing in birds.

Recent developments in cochlear physiology.

Recent findings in cochlear physiology have caused many of our long held ideas about how sound is analyzed by the ear to be reevaluated. This article describes changes which have occurred in three classical ideas of cochlear transduction: (1) There is a gradient of frequency representation along the cochlea with high frequencies being represented at the base and lower frequencies represented progressively toward the apex. It is now known that the specific frequency which is represented at a given location along the cochlea is not invariant but changes systematically during the normal development of hearing. (2) The place code and frequency tuning along the cochlea are due to the conventional traveling wave of von Békésy and basilar membrane mechanics. Experiments in nonmammalian vertebrates which lack a traveling wave have shown that other mechanisms, including the mechanical resonance of hair cell stereocilia, may contribute to tonotopic organization and frequency tuning. It is possible that hair cell stereocilia also contribute to frequency representation and tuning in the mammalian cochlea. (3) The vibration of the basilar membrane to sound is determined by its passive mechanical properties. It is now known that the response of the basilar membrane, and that of the cochlear partition as a whole, is influenced by physiological processes which utilize metabolic energy. The active processes are likely expressed through the motile activity of outer hair cells.

Development of the place principle.

Two experiments using embryonic and hatchling chickens examined how the representation of frequency along the basilar membrane changed during hearing development. In experiment 1, chicks were exposed to high intensity pure tones (500, 1,500, or 3,000 Hz) at one of three different ages. Analysis of hair cell degeneration indicated a discrete region of damage which systematically changed as a function of exposure frequency and age. With maturation, each frequency produced damage at progressively more apical locations. In experiment 2, the representation of frequency in the brain stem auditory nuclei was compared in embryonic, hatchling, and adult chickens. Microelectrode recordings indicated a systematic shift in the frequency representation. Neurons, which are activated by high frequencies in the adult, initially respond to only low frequencies. These experiments indicate how the mature pattern of frequency representation along the basilar membrane gradually emerges during the stages of hearing development.

The effect of unilateral basilar papilla removal upon nuclei laminaris and magnocellularis of the chick examined with [3H]2-deoxy-D-glucose autoradiography.

The effect of unilateral basilar papilla removal on glucose uptake in the 2nd and 3rd order auditory nuclei in the chick's brain stem, nucleus magnocellularis and nucleus laminaris, respectively, was examined with [3H]2-deoxy-D-glucose (2-DG) autoradiography. The tissue was processed according to a thaw-mount technique, and the number of grains in the resulting autoradiographs was counted to assess changes in glucose uptake. It was observed that there is a greater density of grains over the neuropil regions of nucleus laminaris which receive input from the normal ear than over the corresponding regions which receive input from the operated ear. Similarly, differences in grain density are found between the normally innervated and deafferented magnocellular nuclei although these differences are not as great as those in nucleus laminaris. Differences in grain density were also apparent between the glial/fiber regions which bound the neuropil areas of nucleus laminaris; there is a greater density of grains overlying those glial/fiber regions through which fibers receiving input from the normal ear course than over those regions through which fibers which normally carry input from the operated ear travel. It is likely that this difference mainly reflects glucose uptake in the fibers although a possible contribution of glial tissue cannot be excluded. All these effects of basilar papilla removal are seen with survival times as short as 70 min and thus likely reflect the reduction of neural activity rather than the degeneration of pre- or postsynaptic elements. Finally, the same pattern of results as described above was found when using the more common [14C]2-DG procedure or when using [3H]2-DG but processing the tissue using the freeze-dried technique. The present results thus show the neuropil regions of nucleus laminaris and the adjacent glial/fiber areas to be areas of high glucose utilization. Unilateral basilar papilla removal results in the removal of an excitatory input to these regions, and this results in a reduction of glucose utilization that is specific to those neuropil regions and glial/fiber areas that receive input from the operated ear. These findings are contrasted with another study in which removal of a major excitatory input to the dentate gyrus of the rat results in a reduced glucose utilization which is not specific to the deafferented region and which largely reflects post- rather than presynatic events.