Onset Of Hearing Research Articles

Hearing Loss Alters the Subcellular Distribution of Presynaptic GAD and Postsynaptic GABAA Receptors in the Auditory Cortex

We have shown previously that auditory experience regulates the maturation of excitatory synapses in the auditory cortex (ACx). In this study, we used electron microscopic immunocytochemistry to determine whether the heightened excitability of the ACx following neonatal sensorineural hearing loss (SNHL) also involves pre- or postsynaptic alterations of GABAergic synapses. SNHL was induced in gerbils just prior to the onset of hearing (postnatal day 10). At P17, the gamma-aminobutyri acid type A (GABA(A)) receptor's beta2/3-subunit (GABA(A)beta2/3) clusters residing at plasma membranes in layers 2/3 of ACx was reduced significantly in size (P < 0.05) and number (P < 0.005), whereas the overall number of immunoreactive puncta (intracellular + plasmalemmal) remained unchanged. The reduction of GABA(A)beta2/3 was observed along perikaryal plasma membranes of excitatory neurons but not of GABAergic interneurons. This cell-specific change can contribute to the enhanced excitability of SNHL ACx. Presynaptically, GABAergic axon terminals were significantly larger but less numerous and contained 47% greater density of glutamic acid decarboxylase immunoreactivity (P < 0.05). This suggests that GABA synthesis may be upregulated by a retrograde signal arising from lowered levels of postsynaptic GABA(A)R. Thus, both, the pre- and postsynaptic sides of inhibitory synapses that form upon pyramidal neurons of the ACx are regulated by neonatal auditory experience.

Dynamic development of the calyx of Held synapse

The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held.

Inhibitory synaptogenesis in the rat anteroventral cochlear nucleus

Spherical cells in the anteroventral division of the cochlear nucleus, which relay excitatory inputs from the auditory nerve, also receive both GABAergic and glycinergic inhibitory synapses. Inhibition mediated by GABA and glycine fulfils essential roles in the processing abilities of these and other auditory neurons. However, the developmental program leading to a mature complement of GABAergic and glycinergic synapses and microcircuits is largely unknown. Because of their relatively simple geometry, spherical cells provide an excellent model for unraveling basic developmental patterns of inhibitory synaptogenesis. Using a combination of high resolution immunocytochemical methods, we report that, in the rat, synapses containing GABA or glycine are deployed on spherical cell bodies over a time period extending well beyond hearing onset. Such postnatal developmental recruitment of inhibitory endings is progressive, although there are two distinct leaps in their numbers. The first occurs by the end of the first postnatal week, prior to hearing onset, and the second, during the third postnatal week, after hearing onset. This pattern suggests that adjustments in inhibition could be driven by acoustic experience. While GABAergic and glycinergic endings are maturing and growing in number and size, their neurotransmitter content also appears to be developmentally regulated. Quantitative ultrastructural immunocytochemistry with colloidal gold suggests that GABA and glycine accumulation in synaptic endings follows a staggered pattern, with labeling stabilizing at adult levels by postnatal day 21. This may account for adjustments in synaptic efficacy and strength.

Calcium- and Calmodulin-Dependent Inactivation of Calcium Channels in Inner Hair Cells of the Rat Cochlea

Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.

Bilateral cochlear ablation in postnatal rat disrupts development of banded pattern of projections from the dorsal nucleus of the lateral lemniscus to the inferior colliculus

Axonal projections from the dorsal nucleus of the lateral lemniscus (DNLL) distribute contralaterally in a pattern of banded layers in the central nucleus of the inferior colliculus (IC). The banded pattern of DNLL projections is already in the IC by onset of hearing in postnatal rat pups. Previously, it was shown that unilateral cochlear ablation in neonatal rat pups disrupted the banded pattern in IC for the projections of the DNLL contralateral to the ablation but not those of the DNLL ipsilateral to the ablation. In the present study, bilateral cochlear ablation or sham surgery was performed at postnatal day 9 (P9) after which rat pups were killed at P12 and the brains removed to study axonal projections of the DNLL. A lipophilic carbocyanine dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), was placed in the dorsal tegmental commissure of Probst to label decussating DNLL axons that end in the central nucleus of the contralateral IC. The distribution of labeled fibers across the central nucleus of the IC was analyzed in digital images by comparing the pattern of labeling with a sine model of periodic distribution of banded layers. In the control group, labeled axons formed a regular pattern of dense banded layers in IC. In the bilateral cochlear ablation group, labeled axons in the IC were distributed diffusely and there was little or no regular pattern of dense bands of axonal labeling. The influence of the cochlea on developing auditory circuits possibly mediated by activity-dependent mechanisms is discussed.

Effects of omni-directional noise-exposure during hearing onset and age on auditory spatial resolution in the Mongolian gerbil ( Meriones unguiculatus) — a behavioral approach

Inhibitory inputs to the binaural brainstem nuclei medial and lateral superior olives (MSO and LSO, respectively) are thought to be important for sound localization in mammals. Here, we investigate whether aged gerbils that typically exhibit degenerative changes in auditory nuclei providing inhibition to MSO and LSO show diminished localization ability. We also tested the localization ability in gerbils reared in omni-directional white noise during hearing onset, a treatment that affects the adjustment of inhibitory inputs to MSO neurons possibly resulting in weakened sensitivity to interaural time difference. Localization ability of both groups was compared to that of young gerbils raised under control conditions. Stimuli had a duration of 125 ms and were pure tones of 0.5, 1, 2, 4 and 8 kHz, 300-Hz-bands of noise centered at 0.5, 2 and 8 kHz or broad-band noise. Gerbils trained in a two-alternative-forced-choice procedure indicated if sounds were presented from the left or from the right by choosing the respective response compartment of a Y-shaped experimental setup. The minimum resolvable angle (MRA) was calculated as the minimum angle between two loudspeaker locations that a gerbil was able to discriminate. MRAs for aged gerbils were higher compared to controls, whereas MRAs of noise-reared gerbils did not differ from those of the control group. Results are discussed with respect to the progressive degeneration affecting the gerbil's auditory system, changes in the anatomical arrangement of inhibitory inputs on binaural neurons in the MSO, and hearing thresholds.

The Ca V3.1 T-type Ca 2+channel contributes to voltage-dependent calcium currents in rat outer hair cells

Calcium currents through voltage-dependent Ca 2+channels (VDCCs) in mammalian outer hair cells (OHCs) are generally considered to possess the pharmacological properties of L-type (dihydropyridine-sensitive) currents. However, the OHCs' low resting potentials and their slight depolarization upon sound stimuli suggest that the low voltage-activated channels may contribute to Ca 2+regulation. We present morphological and electrophysiological evidence for the presence of the Ca V3.1 T-type Ca 2+channels, one of the low voltage-activated Ca 2+channels, in mature rat OHCs. PCR experiments revealed the expression of Ca V3.1, but not Ca V3.2 or Ca V3.3, in the mature rat cochlea. In situ hybridization and immunohistochemistry revealed expression of Ca V3.1 in both inner and outer hair cells at the mRNA level, but only in the OHCs at the protein level. Western blot analysis of anti-Ca V3.1 antibody showed a 242 kDa band in mature rat cochlear lysates. Patch-clamp recordings of OHCs isolated from rat cochleae after the onset of hearing revealed that whole-cell voltage-dependent Ca 2+ currents were significantly increased in depolarizing steps from a holding potential of − 100 mV when compared with those from − 70 mV. Only the currents from − 100 mV manifested a distinct transient inward Ca 2+ current, and this transient component was effectively blocked by 1 μM of the T-type-specific antagonist, mibefradil. Our data suggest an involvement of Ca V3.1 in intracellular Ca 2+regulation in mature OHCs.

Sensorineural Deafness and Seizures in Mice Lacking Vesicular Glutamate Transporter 3

The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.

Developmental Changes of Mechanics Measured in the Gerbil Cochlea

This report describes stiffness and best frequency measurements obtained in vitro from the basilar membrane of the gerbil cochlea at the onset of hearing, during hearing maturation, and after hearing has matured. Our stiffness data constitute the first direct experimental evidence of developmental stiffness changes in the basal and middle turns. Stiffness changes by a factor of 5.5 in the basal turn between postnatal day 11 and adult, and the difference from adult is statistically significant for all ages measured up to postnatal day 16. For the middle turn, stiffness changes by a factor of 1.6 between postnatal day 11 and adult. Whereas for postnatal day 12 and beyond there is no statistically significant difference from adult, our data suggest that there may be a significant difference of stiffness between day 11 and adult in the middle turn. For the basal turn, our motion measurements confirm a passive component to the developmental best frequency shift. For the middle turn, changes in best frequency are not statistically significant. Best frequency was determined by stimulating the tissue at audio frequencies with a glass paddle and measuring motion with a computer-based imaging system. Tissue stiffness was measured with a piezoelectric-based sensor system. Tissue stiffness changes have previously been postulated to contribute to the best frequency shift observed in the cochlear base. Incorporating our data into a simple spring-mass resonance model demonstrates that our experimentally measured stiffness change can account for the change of best frequency. These results suggest that a stiffness change is, in fact, a critical component of the best frequency shift observed in the basal turn of the gerbil cochlea after the onset of hearing.

The origin of spontaneous activity in the developing auditory system

Spontaneous activity in the developing auditory system is required for neuronal survival as well as the refinement and maintenance of tonotopic maps in the brain. However, the mechanisms responsible for initiating auditory nerve firing in the absence of sound have not been determined. Here we show that supporting cells in the developing rat cochlea spontaneously release ATP, which causes nearby inner hair cells to depolarize and release glutamate, triggering discrete bursts of action potentials in primary auditory neurons. This endogenous, ATP-mediated signalling synchronizes the output of neighbouring inner hair cells, which may help refine tonotopic maps in the brain. Spontaneous ATP-dependent signalling rapidly subsides after the onset of hearing, thereby preventing this experience-independent activity from interfering with accurate encoding of sound. These data indicate that supporting cells in the organ of Corti initiate electrical activity in auditory nerves before hearing, pointing to an essential role for peripheral, non-sensory cells in the development of central auditory pathways.

Synaptic Vesicle Endocytosis at a CNS Nerve Terminal: Faster Kinetics at Physiological Temperatures and Increased Endocytotic Capacity During Maturation

Synaptic vesicle membrane must be quickly retrieved and recycled after copious exocytosis to limit the depletion of vesicle pools. The rate of endocytosis at the calyx of Held nerve terminal has been measured directly using membrane capacitance measurements from immature postnatal day P7-P10 rat pups at room temperature (RT: 23-24 degrees C). This rate has an average time constant of tens of seconds and becomes slower when the amount of exocytosis (measured as capacitance jump) increases. Such slow rates seem paradoxical for a synapse that can operate continuously at high-input frequencies. Here we perform time-resolved membrane capacitance measurements from the mouse calyx of Held in brain stem slices at physiological temperature (PT: 35-37 degrees C), and also from more mature calyces after the onset of hearing (P14-P18). Our results show that the rate of endocytosis is strongly temperature dependent, whereas the endocytotic capacity of a nerve terminal is dependent on developmental stage. At PT we find that endocytosis accelerates due to the addition of a kinetically fast component (time constant: tau = 1-2 s) immediately after exocytosis. Surprisingly, we find that at RT the rate of endocytosis triggered by short (1- to 5-ms) or long (> or =10-ms) depolarizing pulses in P14-P18 mice are similar (tau approximately 15 s). Furthermore, this rate is greatly accelerated at PT (tau approximately 2 s). Thus endocytosis becomes faster and less saturable during synaptic maturation, making the calyceal terminal more capable of sustaining prolonged high-frequency transmitter release.

Spontaneous Discharge Patterns in Cochlear Spiral Ganglion Cells Before the Onset of Hearing in Cats

Spontaneous neural activity has been recorded in the auditory nerve of cats as early as 2 days postnatal (P2), yet individual auditory neurons do not respond to ambient sound levels <90-100 dB SPL until about P10. Significant refinement of the central projections from the spiral ganglion to the cochlear nucleus occurs during this neonatal period. This refinement may be dependent on peripheral spontaneous discharge activity. We recorded from single spiral ganglion cells in kittens aged P3-P9. The spiral ganglion was accessed through the round window through the spiral lamina. A total of 112 ganglion cells were isolated for study in nine animals. Spike rates in neonates were very low, ranging from 0.06 to 56 spikes/s, with a mean of 3.09 +/- 8.24 spikes/s. Ganglion cells in neonatal kittens exhibited remarkable repetitive spontaneous bursting discharge patterns. The unusual patterns were evident in the large mean interval CV (CV(i) = 2.9 +/- 1.6) and burst index of 5.2 +/- 3.5 across ganglion cells. Spontaneous bursting patterns in these neonatal mammals were similar to those reported for cochlear ganglion cells of the embryonic chicken, suggesting this may be a general phenomenon that is common across animal classes. Rhythmic spontaneous discharge of retinal ganglion cells has been shown to be important in the development of central retinotopic projections and normal binocular vision. Bursting rhythms in cochlear ganglion cells may play a similar role in the auditory system during prehearing periods.

Development of gerbil medial superior olive: integration of temporally delayed excitation and inhibition at physiological temperature.

The sensitivity of medial superior olive (MSO) neurons to tens of microsecond differences in interaural temporal delay (ITD) derives in part from their membrane electrical characteristics, kinetics and timing of excitatory and inhibitory inputs, and dendrite structure. However, maturation of these physiological and structural characteristics are little studied, especially in relationship to the onset of auditory experience. We showed, using brain slices at physiological temperature, that MSO neurons exhibited sensitivity to simulated temporally delayed (TD) EPSCs (simEPSC), injected through the recording electrode, by the initial phase of hearing onset at P10, and TD sensitivity was reduced by block of low threshold potassium channels. The spike generation mechanism matured between P10 and P16 to support TD sensitivity to adult-like excitatory stimuli (1-4 ms duration) by P14. IPSP duration was shorter at physiological temperature than reported for lower temperatures, was longer than EPSP duration at young ages, but approached the duration of EPSPs by P16, when hearing thresholds neared maturity. Dendrite branching became less complex over a more restricted time frame between P10 and P12. Because many physiological and structural properties approximated mature values between P14 and P16, we studied temporal integration of simEPSCs and IPSPs at P15. Only a narrow range of relative onset times (< 1 ms) yielded responses showing sensitivity to TD. We propose that shaping of excitatory circuitry to mediate TD sensitivity can begin before airborne sound is detectable, and that inhibitory inputs having suboptimal neural delays may then be pruned by cellular mechanisms activated by sensitivity to ITD.

OPA1, the disease gene for optic atrophy type Kjer, is expressed in the inner ear

Autosomal dominant optic atrophy (adOA) is the most common form of hereditary optic neuropathy. The majority of cases are associated with mutations in the OPA1 gene. A few cases of adOA are known to be associated with moderate progressive hearing loss. To gain insight into the pathogenesis of this hearing loss, we performed expression analyses of OPA1 in the rat auditory and vestibular organ. In cochlear tissue, several splice variants of OPA1 were detected, which are also expressed in retinal tissue. OPA1 mRNA and protein was found in the hair cells and ganglion cells of the cochlea and vestibular organ. In ganglion cells, OPA1 mRNA and protein was already detectable at birth, whereas in the organ of Corti OPA1 mRNA and protein was up-regulated after birth and reached mature-like expression level during the onset of hearing. Comparison of an antibody directed to the mitochondrial marker protein HSP60 with antibodies directed to different amino acid stretches of OPA1 revealed a sub-cellular distribution of OPA1 in areas of significant density of mitochondria. The data suggest that defects in OPA1 cause hearing disorders due to a progressing metabolic disturbance of hair and ganglion cells in the inner ear.

The critical period

The critical period

Genetic deletion of SK2 channels in mouse inner hair cells prevents the developmental linearization in the Ca2+ dependence of exocytosis

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+) action potentials (APs) only before the onset of hearing. Although a role for APs in the developing auditory system has not been determined it could, by analogy with other sensory systems, guide the functional maturation of the cochlea before experience-driven activity begins. Spontaneous APs in immature IHCs are shaped by a variety of ion channels including that of the small conductance Ca(2+)-activated K(+) current (SK2), which is only transiently expressed in immature cells. Using SK2 knockout mice we found that SK2 channels are not required for generating APs but are essential for sustaining continuous repetitive spontaneous AP activity in pre-hearing IHCs. Therefore we used this mutant mouse as a model to study possible developmental implications of disrupted AP activity. Immature mutant IHCs showed impaired exocytotic responses, which are likely to be due to the expression of fewer Ca(2+) channels. Exocytosis was also impaired in adult mutant IHCs, although in this case it resulted from a reduced Ca(2+) efficiency and increased Ca(2+) dependence of the synaptic machinery. Since SK2 channels can only have a functional influence on IHCs during immature development and are not directly involved in neurotransmitter release, the altered Ca(2+) dependence of exocytosis in adult IHCs is likely to be a consequence of their disrupted AP activity at immature stages.

Analysis of connexin subunits required for the survival of vestibular hair cells

Mutations in connexin (Cx) genes are responsible for a large proportion of human inherited prelingual deafness cases. The most commonly found human Cx mutations are either Cx26 or Cx30 deletions. Histological observations made in the organ of Corti of homozygous Cx26 and Cx30 gene knockout mice show that cochlear hair cells degenerate after the onset of hearing. However, it is unclear whether vestibular hair cells undergo similar degeneration in connexin knockout mice. To address this question, we first examined expression patterns of Cx26 and Cx30 in the saccule, utricle, and ampulla by immunolabeling. In wild-type mice, Cx26 and Cx30 immunoreactivity was found extensively in vestibular supporting cells and connective tissue cells, and the two Cxs were co-localized in most gap junction (GJ) plaques. Targeted deletion of the Cx30 gene, which caused little change in Cx26 expression pattern, resulted in a significant and age-related loss of vestibular hair cells only in the saccule. dUTP nick end labeling (TUNEL) staining also revealed on-going apoptosis specifically in saccular hair cells of Cx30(-/-) mice. These results indicated that hair cell survival in the utricle and ampulae does not require Cx30. Importantly, over-expressing the Cx26 gene from a modified bacterial artificial chromosome in the Cx30(-/-) background rescued the saccular hair cells. These results suggest that it is the reduction in the total amount of GJs rather than the specific loss of Cx30 that underlies saccular hair cell death in Cx30(-/-) mice. Hybrid GJs co-assembled from Cx26 and Cx30 were not essential for the survival of saccular hair cells.

Spontaneous Generation in Early Sensory Development. Focus on “Spontaneous Discharge Patterns in Cochlear Spiral Ganglion Cells Before the Onset of Hearing in Cats”

Sensory systems are our gateways to the outside world. How are these complex systems—sensory organ and the associated brain pathways—set up during development? Molecular cues are of course essential in establishing many basic cellular characteristics and the connections between all elements of a

Early segregation of layered projections from the lateral superior olivary nucleus to the central nucleus of the inferior colliculus in the neonatal cat

The central nucleus of the inferior colliculus (IC) is a laminated structure that receives multiple converging afferent projections. These projections terminate in a layered arrangement and are aligned with dendritic arbors of the predominant disc-shaped neurons, forming fibrodendritic laminae. Within this structural framework, inputs terminate in a precise manner, establishing a mosaic of partially overlapping domains that likely define functional compartments. Although several of these patterned inputs have been described in the adult, relatively little is known about their organization prior to hearing onset. The present study used the lipophilic carbocyanine dyes DiI and DiD to examine the ipsilateral and contralateral projections from the lateral superior olivary (LSO) nucleus to the IC in a developmental series of paraformaldehyde-fixed kitten tissue. By birth, the crossed and uncrossed projections had reached the IC and were distributed across the frequency axis of the central nucleus. At this earliest postnatal stage, projections already exhibited a characteristic banded arrangement similar to that described in the adult. The heaviest terminal fields of the two inputs were always complementary in nature, with the ipsilateral input appearing slightly denser. This early arrangement of interdigitating ipsilateral and contralateral LSO axonal bands that occupy adjacent sublayers supports the idea that the initial establishment of this highly organized mosaic of inputs that defines distinct synaptic domains within the IC occurs largely in the absence of auditory experience. Potential developmental mechanisms that may shape these highly ordered inputs prior to hearing onset are discussed.

Large-scale reorganization of the tonotopic map in mouse auditory midbrain revealed by MRI

The cortex is thought to be the primary site of sensory plasticity, particularly during development. Here, we report that large-scale reorganization of the mouse auditory midbrain tonotopic map is induced by a specific sound-rearing environment consisting of paired low- (16 kHz) and high-frequency (40 kHz) tones. To determine the potential for plasticity in the mouse auditory midbrain, we used manganese-enhanced MRI to analyze the midbrain tonotopic maps of control mice during normal development and mice reared in the two-tone (16 + 40 kHz) environment. We found that the tonotopic map emerged during the third postnatal week in normal mice. Before 3 weeks, a larger percentage of auditory midbrain responded to each of the suprathreshold test frequencies, despite the fact that the primary afferent projections are in place even before hearing onset. By 3 weeks, the midbrain tonotopic map of control mice was established, and manganese-enhanced MRI showed a clear separation between the 16- and 40-kHz responses. Two-tone rearing dramatically altered the appearance of these discrete frequency-specific responses. A significant volume of the auditory midbrain became responsive to both rearing frequencies, resulting in a large-scale reorganization of the tonotopic map. These results indicate that developmental plasticity occurs on a much greater scale than previously appreciated in the mammalian auditory midbrain.