Auditory Research publications

Spiral ganglion neuron (SGN) damage and apoptosis can lead to noise-induced hearing loss, age-associated hearing loss and, in certain cases, auditory neuropathy. The apoptosis-inducing factor (AIF)-associated pathway may be important in this process. The present study aimed to investigate the expression levels of AIF and calpain in damaged SGNs. Glutamate (Glu) perfusion and cell culture in different concentrations of Glu were performed to damage the SGNs of Sprague-Dawley (SD) rats, with saline water used as a control Different concentrations (5, 10, 20 and 40 mM) of Glu were injected into the cochlear tympanic canal of 18 SD rats, and 10, 20 and 40 mM Glu were added to SGN cultures. Auditory brainstem responses (ABR) were measured prior to and 2 days following the injection of Glu. Immunofluorescent staining was used to detect the SGN damage and the expression levels of AIF and calpain in vivo and in in vitro. Transmission electron microscopy (TEM) was used to measure cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to analyse the gene expression levels of AIF and calpain in the damaged SGNs. The TEM identified mitochondrial vacuolisation, swelling of the SGN and heterochromatin formation. Injection of Glu reduced the number of SGNs and induced apoptosis. AIF was observed to translocate into the nuclei of the SGNs in the 20 and 40 mM Glu groups, and the expression levels of AIF and calpain were markedly upregulated in the modiolus of the Glu-damaged SGNs. The upregulation of AIF and calpain may be important in the process of SGN damage and apoptosis.

In auditory cortex, temporal information within a sound is represented by two complementary neural codes: a temporal representation based on stimulus-locked firing and a rate representation, where discharge rate co-varies with the timing between acoustic events but lacks a stimulus-synchronized response. Using a computational neuronal model, we find that stimulus-locked responses are generated when sound-evoked excitation is combined with strong, delayed inhibition. In contrast to this, a non-synchronized rate representation is generated when the net excitation evoked by the sound is weak, which occurs when excitation is coincident and balanced with inhibition. Using single-unit recordings from awake marmosets (Callithrix jacchus), we validate several model predictions, including differences in the temporal fidelity, discharge rates and temporal dynamics of stimulus-evoked responses between neurons with rate and temporal representations. Together these data suggest that feedforward inhibition provides a parsimonious explanation of the neural coding dichotomy observed in auditory cortex.

Sound localization is an important function of the human brain, but the underlying cortical mechanisms remain unclear. In this study, we recorded auditory stimuli in three-dimensional space and then replayed the stimuli through earphones during functional magnetic resonance imaging (fMRI). By employing a machine learning algorithm, we successfully decoded sound location from the blood oxygenation level-dependent signals in the temporal lobe. Analysis of the data revealed that different cortical patterns were evoked by sounds from different locations. Specifically, discrimination of sound location along the abscissa axis evoked robust responses in the left posterior superior temporal gyrus (STG) and right mid-STG, discrimination along the elevation (EL) axis evoked robust responses in the left posterior middle temporal lobe (MTL) and right STG, and discrimination along the ordinate axis evoked robust responses in the left mid-MTL and right mid-STG. These results support a distributed representation of acoustic space in human cortex.

Amplitude modulations are fundamental features of natural signals, including human speech and nonhuman primate vocalizations. Because natural signals frequently occur in the context of other competing signals, we used a forward-masking paradigm to investigate how the modulation context of a prior signal affects cortical responses to subsequent modulated sounds. Psychophysical “modulation masking,” in which the presentation of a modulated “masker” signal elevates the threshold for detecting the modulation of a subsequent stimulus, has been interpreted as evidence of a central modulation filterbank and modeled accordingly. Whether cortical modulation tuning is compatible with such models remains unknown. By recording responses to pairs of sinusoidally amplitude modulated (SAM) tones in the auditory cortex of awake squirrel monkeys, we show that the prior presentation of the SAM masker elicited persistent and tuned suppression of the firing rate to subsequent SAM signals. Population averages of these effects are compatible with adaptation in broadly tuned modulation channels. In contrast, modulation context had little effect on the synchrony of the cortical representation of the second SAM stimuli and the tuning of such effects did not match that observed for firing rate. Our results suggest that, although the temporal representation of modulated signals is more robust to changes in stimulus context than representations based on average firing rate, this representation is not fully exploited and psychophysical modulation masking more closely mirrors physiological rate suppression and that rate tuning for a given stimulus feature in a given neuron's signal pathway appears sufficient to engender context-sensitive cortical adaptation.

Various factors that are important for proper hearing have been identified, including serum levels of zinc. Here we investigated whether epigenetic regulatory pathways, which can be modified by environmental factors, could modulate hearing. RT-PCR detected expression of genes encoding DNA methyltransferase and histone deacetylase (Hdac) in the postnatal as well as adult mouse auditory epithelium. DBA/2J mice, which are a model for progressive hearing loss, were injected subcutaneously with one or a combination of the following reagents: <smallcaps>L</smallcaps>-methionine as a methyl donor, valproic acid as a pan-Hdac inhibitor, and folic acid and vitamin B12 as putative factors involved in age-related hearing loss. The mice were treated from ages 4 to 12 weeks (N ≥ 5), and auditory brainstem response (ABR) thresholds were measured at 8, 16, and 32 kHz. Treatment of the mice with a combination of <smallcaps>L</smallcaps>-methionine and valproic acid (M+V) significantly reduced the increase in the ABR threshold at 32 kHz. Treatment with any of these reagents individually produced no such effect. Microarray analyses detected 299 gene probes that were significantly up- or down-regulated in the cochleae of mice treated with M+V compared with the control vehicle-treated mice. Quantitative RT-PCR confirmed significant up-regulation of a zinc importer gene, Zip4, in the cochleae of mice treated with M+V. Immunohistochemistry demonstrated an intense Zip4 signal in cochlear tissues such as the lateral wall, organ of Corti, and spiral ganglion. Finally, mice treated with the Zip4 inducer (-)-epigallocatechin-3-O-gallate showed a significant reduction in the increase of the ABR threshold at 32 kHz and up-regulation of Zip4 expression in the cochlea. This study suggests that epigenetic regulatory pathways can modify auditory function and that zinc intake in the cochlea via Zip4 mediates maintenance of mammalian hearing.

Congenital human cytomegalovirus (HCMV) occurs in 0.5-1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD(3+) mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.

High dose sodium salicylate causes moderate, reversible hearing loss and tinnitus. Salicylate-induced hearing loss is believed to arise from a reduction in the electromotile response of outer hair cells (OHCs) and/or reduction of KCNQ4 potassium currents in OHCs, which decreases the driving force for the transduction current. Therefore, enhancing OHC potassium currents could potentially prevent salicylate-induced temporary hearing loss. In this study, we tested whether opening voltage-gated potassium channels using ICA-105665, a novel small molecule that opens KCNQ2/3 and KCNQ3/5 channels, can reduce salicylate-induced hearing loss. We found that systemic application of ICA-105665 at 10 mg/kg prevented the salicylate-induced amplitude reduction and threshold shift in the compound action potentials recorded at the round window of the cochlea. ICA-105665 also prevented the salicylate-induced reduction of distortion-product otoacoustic emission. These results suggest that ICA-105665 partially compensates for salicylate-induced cochlear hearing loss by enhancing KCNQ2/3 and KCNQ3/5 potassium currents and the motility of OHCs.

This study compared the timing of appearance of three components of age-related hearing loss that determine the pattern and severity of presbycusis: the functional and structural pathologies of sensory cells and neurons and changes in gap detection (GD), the latter as an indicator of auditory temporal processing. Using UM-HET4 mice, genetically heterogeneous mice derived from four inbred strains, we studied the integrity of inner and outer hair cells by position along the cochlear spiral, inner hair cell-auditory nerve connections, spiral ganglion neurons (SGN), and determined auditory thresholds, as well as pre-pulse and gap inhibition of the acoustic startle reflex (ASR). Comparisons were made between mice of 5-7, 22-24 and 27-29 months of age. There was individual variability among mice in the onset and extent of age-related auditory pathology. At 22-24 months of age a moderate to large loss of outer hair cells was restricted to the apical third of the cochlea and threshold shifts in the auditory brain stem response were minimal. There was also a large and significant loss of inner hair cell-auditory nerve connections and a significant reduction in GD. The expression of Ntf3 in the cochlea was significantly reduced. At 27-29 months of age there was no further change in the mean number of synaptic connections per inner hair cell or in GD, but a moderate to large loss of outer hair cells was found across all cochlear turns as well as significantly increased ABR threshold shifts at 4, 12, 24 and 48 kHz. A statistical analysis of correlations on an individual animal basis revealed that neither the hair cell loss nor the ABR threshold shifts correlated with loss of GD or with the loss of connections, consistent with independent pathological mechanisms.

Speaking is one of the most complex motor behaviors developed to facilitate human communication. The underlying neural mechanisms of speech involve sensory-motor interactions that incorporate feedback information for online monitoring and control of produced speech sounds. In the present study, we adopted an auditory feedback pitch perturbation paradigm and combined it with functional magnetic resonance imaging (fMRI) recordings in order to identify brain areas involved in speech production and motor control. Subjects underwent fMRI scanning while they produced a steady vowel sound /a/ (speaking) or listened to the playback of their own vowel production (playback). During each condition, the auditory feedback from vowel production was either normal (no perturbation) or perturbed by an upward (+600 cents) pitch-shift stimulus randomly. Analysis of BOLD responses during speaking (with and without shift) vs. rest revealed activation of a complex network including bilateral superior temporal gyrus (STG), Heschl's gyrus, precentral gyrus, supplementary motor area (SMA), Rolandic operculum, postcentral gyrus and right inferior frontal gyrus (IFG). Performance correlation analysis showed that the subjects produced compensatory vocal responses that significantly correlated with BOLD response increases in bilateral STG and left precentral gyrus. However, during playback, the activation network was limited to cortical auditory areas including bilateral STG and Heschl's gyrus. Moreover, the contrast between speaking vs. playback highlighted a distinct functional network that included bilateral precentral gyrus, SMA, IFG, postcentral gyrus and insula. These findings suggest that speech motor control involves feedback error detection in sensory (e.g. auditory) cortices that subsequently activate motor-related areas for the adjustment of speech parameters during speaking.

Understanding the principles by which the brain combines information from different senses provides us with insight into the computational strategies used to maximize their utility. Prior studies of the superior colliculus (SC) neuron as a model suggest that the relative timing with which sensory cues appear is an important factor in this context. Cross-modal cues that are near-simultaneous are likely to be derived from the same event, and the neural inputs they generate are integrated more strongly than those from cues that are temporally displaced from one another. However, the present results from studies of cat SC neurons show that this “temporal principle” of multisensory integration is more nuanced than previously thought and reveal that the integration of temporally displaced sensory responses is also highly dependent on the relative efficacies with which they drive their common target neuron. Larger multisensory responses were achieved when stronger responses were advanced in time relative to weaker responses. This new temporal principle of integration suggests an inhibitory mechanism that better accounts for the sensitivity of the multisensory product to differences in the timing of cross-modal cues than do earlier mechanistic hypotheses based on response onset alignment or response overlap.

The central representation of a given acoustic motif is thought to be strongly context dependent, i.e., to rely on the spectrotemporal past and present of the acoustic mixture in which it is embedded. The present study investigated the cortical representation of spectral edges (i.e., where stimulus energy changes abruptly over frequency) and its dependence on stimulus duration and depth of the spectral contrast in guinea pig. We devised a stimulus ensemble composed of random tone pips with or without an attenuated frequency band (AFB) of variable depth. Additionally, the multitone ensemble with AFB was interleaved with periods of silence or with multitone ensembles without AFB. We have shown that the representation of the frequencies near but outside the AFB is greatly enhanced, whereas the representation of frequencies near and inside the AFB is strongly suppressed. These cortical changes depend on the depth of the AFB: although they are maximal for the largest depth of the AFB, they are also statistically significant for depths as small as 10 dB. Finally, the cortical changes are quick, occurring within a few seconds of stimulus ensemble presentation with AFB, and are very labile, disappearing within a few seconds after the presentation without AFB. Overall, this study demonstrates that the representation of spectral edges is dynamically enhanced in the auditory centers. These central changes may have important functional implications, particularly in noisy environments where they could contribute to preserving the central representation of spectral edges.

Perceptual performance in persons with hearing loss, especially those using devices to restore hearing, is not fully predicted by traditional audiometric measurements designed to evaluate the status of peripheral function. The integrity of auditory brainstem synapses may vary with different forms of hearing loss, and differential effects on the auditory nerve-brain interface may have particularly profound consequences for the transfer of sound from ear to brain. Loss of auditory nerve synapses in ventral cochlear nucleus (VCN) has been reported after acoustic trauma, ablation of the organ of Corti, and administration of ototoxic compounds. The effects of gradually acquired forms deafness on these synapses are less well understood. We investigated VCN gross morphology and auditory nerve synapse integrity in DBA/2J mice with early-onset progressive sensorineural hearing loss. Hearing status was confirmed using auditory brainstem response audiometry and acoustic startle responses. We found no change in VCN volume, number of macroneurons, or number of VGLUT1-positive auditory nerve terminals between young adult and older, deaf DBA/2J. Cell-type specific analysis revealed no difference in the number of VGLUT1 puncta contacting bushy and multipolar cell body profiles, but the terminals were smaller in deaf DBA/2J mice. Transmission electron microscopy confirmed the presence of numerous healthy, vesicle-filled auditory nerve synapses in older, deaf DBA/2J mice. The present results suggest that synapses can be preserved over a relatively long time-course in gradually acquired deafness. Elucidating the mechanisms supporting survival of central auditory nerve synapses in models of acquired deafness may reveal new opportunities for therapeutic intervention.

Sensorineural hearing loss is most commonly caused by the death of hair cells in the organ of Corti, and once lost, mammalian hair cells do not regenerate. In contrast, other vertebrates such as birds can regenerate hair cells by stimulating division and differentiation of neighboring supporting cells. We currently know little of the genetic networks which become active in supporting cells when hair cells die and that are activated in experimental models of hair cell regeneration. Several studies have shown that neonatal mammalian cochlear supporting cells are able to trans-differentiate into hair cells when cultured in conditions in which the Notch signaling pathway is blocked. We now show that the ability of cochlear supporting cells to trans-differentiate declines precipitously after birth, such that supporting cells from six-day-old mouse cochlea are entirely unresponsive to a blockade of the Notch pathway. We show that this trend is seen regardless of whether the Notch pathway is blocked with gamma secretase inhibitors, or by antibodies against the Notch1 receptor, suggesting that the action of gamma secretase inhibitors on neonatal supporting cells is likely to be by inhibiting Notch receptor cleavage. The loss of responsiveness to inhibition of the Notch pathway in the first postnatal week is due in part to a down-regulation of Notch receptors and ligands, and we show that this down-regulation persists in the adult animal, even under conditions of noise damage. Our data suggest that the Notch pathway is used to establish the repeating pattern of hair cells and supporting cells in the organ of Corti, but is not required to maintain this cellular mosaic once the production of hair cells and supporting cells is completed. Our results have implications for the proposed used of Notch pathway inhibitors in hearing restoration therapies.

Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.

Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS) the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL). After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action.

The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear-especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.

Neuromodulation is an increasingly accepted treatment for neurological and psychiatric disorders but is limited by its invasiveness or its inability to target deep brain structures using noninvasive techniques. We propose a new concept called Multimodal Synchronization Therapy (mSync) for achieving targeted activation of the brain via noninvasive and precisely timed activation of auditory, visual, somatosensory, motor, cognitive and limbic pathways. In this initial study in guinea pigs, we investigated mSync using combined activation of just the auditory and somatosensory pathways, which induced differential and timing dependent plasticity in neural firing within deep brain and cortical regions of the auditory system. Furthermore, by varying the location of somatosensory stimulation across the body, we increased or decreased spiking activity across different neurons. These encouraging results demonstrate the feasibility of systematically modulating the brain using mSync. Considering that hearing disorders such as tinnitus and hyperacusis have been linked to abnormal and hyperactive firing patterns within the auditory system, these results open up the possibility for using mSync to decrease this pathological activity by varying stimulation parameters. Incorporating multiple types of pathways beyond just auditory and somatosensory inputs and using other activation patterns may enable treatment of various brain disorders.

Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor β (TGF-β) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-β as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-β1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-β1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-β1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.

The interplay of cortical excitation and inhibition is a fundamental feature of cortical information processing. Excitation and inhibition in single cortical neurons are balanced in their response to optimal sensory stimulation due to thalamocortical feedforward microcircuitry. It is unclear whether the balance between cortical excitation and inhibition is maintained at the threshold stimulus level. Using in vivo whole-cell patch-clamp recording of thalamocortical recipient neurons in the primary auditory cortex of mice, we examined the tone-evoked excitatory and inhibitory postsynaptic currents at threshold levels. Similar to previous reports, tone induced excitatory postsynaptic currents when the membrane potentials were held at 70 mV and inhibitory postsynaptic currents when the membrane potentials were held at 0 mV on single cortical neurons. This coupled excitation and inhibition is not demonstrated when threshold-level tone stimuli are presented. In most cases, tone induced only excitatory postsynaptic current. The best frequencies of excitatory and inhibitory responses were often different and thresholds of inhibitory responses were mostly higher than those of excitatory responses. Our data suggest that the excitatory and inhibitory inputs to single cortical neurons are imbalanced at the threshold level. This imbalance may result from the inherent dynamics of thalamocortical feedforward microcircuitry.

Tinnitus has been suggested to arise from neuronal hyperactivity in auditory areas of the brain, and anti-epileptic drugs are sometimes used to provide relief from tinnitus. Recently, the anti-epileptic properties of the cannabinoid drugs have gained increasing interest; however, the use of cannabinoids as a form of treatment for tinnitus is controversial. In this study, we tested whether a combination of delta-9-tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD), delivered in a 1:1 ratio, could affect tinnitus perception in a rat model of acoustic trauma-induced tinnitus. Following sham treatment or acoustic trauma, the animals were divided into the following groups: (1) sham (i.e., no acoustic trauma) with vehicle treatment; (2) sham with drug treatment (i.e., delta-9-THC + CBD); (3) acoustic trauma-exposed exhibiting tinnitus, with drug treatment; and (4) acoustic trauma-exposed exhibiting no tinnitus, with drug treatment. The animals received either the vehicle or the cannabinoid drugs every day, 30 min before the tinnitus behavioral testing. Acoustic trauma caused a significant increase in the auditory brainstem response (ABR) thresholds in the exposed animals, indicating hearing loss; however, there was a partial recovery over 6 months. Acoustic trauma did not always result in tinnitus; however, among those that did exhibit tinnitus, some of them had tinnitus at multiple frequencies while others had it only at a single frequency. The cannabinoids significantly increased the number of tinnitus animals in the exposed-tinnitus group, but not in the sham group. The results suggest that cannabinoids may promote the development of tinnitus, especially when there is pre-existing hearing damage.