Auditory Research publications

Noise exposure producing temporary threshold shifts (TTS) has been demonstrated to cause permanent changes to cochlear physiology and hearing function. Several explanations have been purported to underlie these long-term changes in cochlear function, such as damage to sensory cell stereocilia and synaptic connections between sensory cells and their innervation by spiral ganglion neurons, and demyelination of the auditory nerve. Though these structural defects have been implicated in hearing difficulty, cochlear responses to this stress damage remains poorly understood. Here, we report the activation of the cochlear immune system following exposure to lower level noise (LLN) that causes only TTS. Using multiple morphological, molecular and functional parameters, we assessed the responses of macrophages, the primary immune cell population in the cochlea, to the LLN exposure. This study reveals that a LLN that causes only TTS increases the macrophage population in cochlear regions immediately adjacent to sensory cells and their innervations. Many of these cells acquire an activated morphology and express the immune molecules CCL2 and ICAM1 that are important for macrophage inflammatory activity and adhesion. However, LLN exposure reduces macrophage phagocytic ability. While the activated morphology of cochlear macrophages reverses, the complete recovery is not achieved 2 months after the LLN exposure. Taken together, these observations clearly implicate the cochlear immune system in the cochlear response to LLN that causes no permanent threshold change.

Auditory perception depends on multi-dimensional information in acoustic signals that must be encoded by auditory nerve fibers (ANF). These dimensions are represented by filters with different frequency selectivities. Multiple models have been suggested; however, the identification of relevant filters and type of interactions has been elusive, limiting progress in modeling the cochlear output. Spike-triggered covariance analysis of barn owl ANF responses was used to determine the number of relevant stimulus filters and estimate the nonlinearity that produces responses from filter outputs. This confirmed that ANF responses depend on multiple filters. The first, most dominant filter was the spike-triggered average, which was excitatory for all neurons. The second and third filters could be either suppressive or excitatory with center frequencies above or below that of the first filter. The nonlinear function mapping the first two filter outputs to the spiking probability ranged from restricted to nearly circular-symmetric, reflecting different modes of interaction between stimulus dimensions across the sample. This shows that stimulus encoding in ANFs of the barn owl is multidimensional and exhibits diversity over the population, suggesting that models must allow for variable numbers of filters and types of interactions between filters to describe how sound is encoded in ANFs.

The brain decomposes mixtures of sounds, such as competing talkers, into perceptual streams that can be attended to individually. Attention can enhance the cortical representation of streams, but it is unknown what acoustic features the enhancement reflects, or where in the auditory pathways attentional enhancement is first observed. Here, behavioral measures of streaming were combined with simultaneous low- and high-frequency envelope-following responses (EFR) that are thought to originate primarily from cortical and subcortical regions, respectively. Repeating triplets of harmonic complex tones were presented with alternating fundamental frequencies. The tones were filtered to contain either low-numbered spectrally resolved harmonics, or only high-numbered unresolved harmonics. The behavioral results confirmed that segregation can be based on either tonotopic or pitch cues. The EFR results revealed no effects of streaming or attention on subcortical responses. Cortical responses revealed attentional enhancement under conditions of streaming, but only when tonotopic cues were available, not when streaming was based only on pitch cues. The results suggest that the attentional modulation of phase-locked responses is dominated by tonotopically tuned cortical neurons that are insensitive to pitch or periodicity cues.

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.

Mutations in GJB2, the gene that encodes connexin 26 (Cx26), are the most common cause of sensorineural hearing impairment. The truncating variant 35delG, which determines a complete loss of Cx26 protein function, is the prevalent GJB2 mutation in several populations. Here, we generated and analyzed Gjb2+/- mice as a model of heterozygous human carriers of 35delG. Compared to control mice, auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) worsened over time more rapidly in Gjb2+/- mice, indicating they were affected by accelerated age-related hearing loss (ARHL), or presbycusis. We linked causally the auditory phenotype of Gjb2+/- mice to apoptosis and oxidative damage in the cochlear duct, reduced release of glutathione from connexin hemichannels, decreased nutrient delivery to the sensory epithelium via cochlear gap junctions and deregulated expression of genes that are under transcriptional control of the nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal regulator of tolerance to redox stress. Moreover, a statistically significant genome-wide association with two genes (PRKCE and TGFB1) related to the Nrf2 pathway (p-value < 4 × 10-2) was detected in a very large cohort of 4091 individuals, originating from Europe, Caucasus and Central Asia, with hearing phenotype (including 1076 presbycusis patients and 1290 healthy matched controls). We conclude that (i) elements of the Nrf2 pathway are essential for hearing maintenance and (ii) their dysfunction may play an important role in the etiopathogenesis of human presbycusis.

Speech perception relies on the accurate resolution of brief, successive sounds that change rapidly over time. Deficits in the perception of such sounds, indicated by a reduced ability to detect signals during auditory backward masking, strongly relate to language processing difficulties in children. Backward masking during normal development has a longer maturational trajectory than many other auditory percepts, implicating the involvement of central auditory neural mechanisms with protracted developmental time courses. Despite the importance of this percept, its neural correlates are not well described at any developmental stage. We therefore measured auditory cortical responses to masked signals in juvenile and adult Mongolian gerbils and quantified the detection ability of individual neurons and neural populations in a manner comparable with psychoacoustic measurements. Perceptually, auditory backward masking manifests as higher thresholds for detection of a short signal followed by a masker than for the same signal in silence. Cortical masking was driven by a combination of suppressed responses to the signal and a reduced dynamic range available for signal detection in the presence of the masker. Both coding elements contributed to greater masked threshold shifts in juveniles compared with adults, but signal-evoked firing suppression was more pronounced in juveniles. Neural threshold shifts were a better match to human psychophysical threshold shifts when quantified with a longer temporal window that included the response to the delayed masker, suggesting that temporally selective listening may contribute to age-related differences in backward masking. NEW & NOTEWORTHY In children, auditory detection of backward masked signals is immature well into adolescence, and detection deficits correlate with problems in speech processing. Our auditory cortical recordings reveal immature backward masking in adolescent animals that mirrors the prolonged development seen in children. This is driven by both signal-evoked suppression and dynamic range reduction. An extended window of analysis suggests that differences in temporally focused listening may contribute to late maturing thresholds for backward masked signals.

The largest targets of retinal input in mammals are the dorsal lateral geniculate nucleus (dLGN), a relay to the primary visual cortex (V1), and the superior colliculus. V1 innervates and influences the superior colliculus. Here, we find that, in turn, superior colliculus modulates responses in mouse V1. Optogenetically inhibiting the superior colliculus reduces responses in V1 to optimally sized stimuli. Superior colliculus could influence V1 via its strong projection to the lateral posterior nucleus (LP/Pulvinar) or its weaker projection to the dLGN. Inhibiting superior colliculus strongly reduces activity in LP. Pharmacologically silencing LP itself, however, does not remove collicular modulation of V1. The modulation is instead due to a collicular gain modulation of the dLGN. Surround suppression operating in V1 explains the different effects for differently sized stimuli. Computations of visual saliency in the superior colliculus can thus influence tuning in the visual cortex via a tectogeniculate pathway.

Echolocating bats measure target distance by the time delay between call and echo. Target movement such as the flutter of insect wings induces delay modulations. Perception of delay modulations has been studied extensively in bats, but only concerning how well bats discriminate flutter frequencies, never with regard to flutter magnitude. We used an auditory virtual reality approach to generate changes in echo delay that were independent of call repetition rate, mimicking fluttering insect wings. We show that in the frequency-modulating (FM) bat Phyllostomus discolor, the sensitivity for modulations in echo delay depends on the rate of the modulation, with bats being most sensitive at modulation rates below 20 Hz and above 50 Hz. The very short duration of their calls compels FM bats to evaluate slow modulations (< about 100 Hz) across entire echo sequences. This makes them susceptible to interference between their own call repetition rate and the modulation rate. We propose that this phenomenon constitutes an echo-acoustic wagon-wheel effect. We further demonstrate how at high modulation rates, flutter sensitivity could be rescued by using spectral and temporal cues introduced by Doppler distortions. Thus, Doppler distortions may play a crucial role in flutter sensitivity in the hundreds of FM species worldwide.

Exposure to heavy metals may lead to hearing impairment. However, experimental studies have not explored this issue with and without noise exposure in mature animals with environmentally relevant doses. The aim of this study was to investigate ototoxicity produced by lead (Pb) and cadmium (Cd) and noise, singly and in combination, in the adult CBA/CaJ mouse. Metals were delivered via drinking water (0.03 mM, 1 mM, and 3 mM Pb; or 30, 100, and 300 μM Cd) for 12 weeks, resulting in environmentally- and occupationally relevant mean (± standard deviations) blood levels of Pb (2.89 ± 0.44, 38.5 ± 4.9, and 60.1 ± 6.6 μg/dl, respectively) and Cd (1.3 ± 0.23, 6.37 ± 0.87, 27.2 ± 4.1 μg/L, respectively). Metal treatment was also combined with a noise exposure consisting of a 105 dB broadband (2-20 kHz) stimulus for 2 hr or a sham exposure. Auditory performance was determined by comparing auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) at baseline and after 11 weeks of metal treatment. Metal-exposed animals did not develop significant auditory deficits and did not exhibit morphological damage to cochlear hair cells. In contrast, noise-exposed animals, including those exposed to combinations of metals and noise, demonstrated significant hair cell loss, reduced DPOAE amplitudes, and ABR threshold shifts of 42.2 ± 13 dB at 32 kHz (105 dB noise alone). No significant potentiation or synergistic effects were found in groups exposed to multiple agents. This study establishes a highly reproducible adult mouse model that may be used to evaluate a variety of environmental exposure mixtures.

Active mechanical amplification of sound occurs in cochlear outer hair cells (OHCs) that change their length with oscillations of their membrane potential. Such length changes are the proposed cellular source of the cochlear amplifier, and prestin is the motor protein responsible for OHC electromotility. Previous findings have shown that mice lacking prestin displayed a loss of OHC electromotility, subsequent loss of distortion-product otoacoustic emissions, and a 40-60 dB increase in hearing thresholds. In this study we were interested in studying the functional consequences of the complete loss of cochlear amplification on neural coding of frequency selectivity, tuning, and temporal processing in the auditory midbrain. We recorded near-field auditory evoked potentials and multi-unit activity from the inferior colliculus (IC) of prestin (-/-) null and prestin (+/+) wild-type control mice and determined frequency response areas (FRAs), tuning sharpness, and gap detection to tone bursts and silent gaps embedded in broadband noise. We were interested in determining if the moderate to severe sensorineural hearing loss associated with the loss of motor protein prestin would also impair auditory midbrain temporal-processing measures, or if compensatory mechanisms within the brainstem could compensate for the loss of prestin. In prestin knockout mice we observed that there are severe impairments in midbrain tuning, thresholds, excitatory drive, and gap detection suggesting that brainstem and midbrain processing could not overcome the auditory processing deficits afforded by the loss of OHC electromotility mediated by the prestin protein.

The auditory brainstem response (ABR) is relatively non-invasive, and in many species, the only practical way to assess auditory sensitivity. The two main methods for measuring ABR are using either transients or tone bursts as a stimulus. The transient stimulus produces strong neural responses that contain no frequency information. In contrast, tone bursts stimulate only a small part of the auditory system, eliciting weaker neural responses but supplying frequency information. Furthermore, short tone bursts become less and less frequency specific with increasing stimulus wavelength, making them unsuitable for testing low-frequency hearing. Here, we develop a method that can measure sensitivity to both low and high-frequency stimuli. The method is based on masking of a transient response by long-duration sinusoids. The measurement system is developed as a highly portable system that runs on battery power. It has been used in a variety of animals in our lab and in the field, including squid (Mooney et al. in J Exp Biol 213: 3748-3759, 2010), lungfish (Christensen-Dalsgaard et al. in J Neurophys 105: 1992-2004, 2011b), alligators (Bierman et al. in J Exp Biol 217: 1094-1107, 2014), and mink (Brandt et al. in J Exp Biol 216: 3542-3550, 2013). Here, we present data recorded from Tokay geckos and compare the results with tone burst ABR measurements. This method produces results comparable to tone burst stimulations at higher frequencies (above 1 kHz) but has several advantages: it is relatively insensitive to fluctuations in neural signal level, it allows measurements at very low frequencies, it allows constant monitoring of the state of the animal, and can be used to measure directional hearing.

Purpose The goal was to evaluate the potential effects of increasing hearing loss and advancing age on spectral envelope perception. Method Spectral modulation detection was measured as a function of spectral modulation frequency from 0.5 to 8.0 cycles/octave. The spectral modulation task involved discrimination of a noise carrier (3 octaves wide from 400 to 3200 Hz) with a flat spectral envelope from a noise having a sinusoidal spectral envelope across a logarithmic audio frequency scale. Spectral modulation transfer functions (SMTFs; modulation threshold vs. modulation frequency) were computed and compared 4 listener groups: young normal hearing, older normal hearing, older with mild hearing loss, and older with moderate hearing loss. Estimates of the internal spectral contrast were obtained by computing excitation patterns. Results SMTFs for young listeners with normal hearing were bandpass with a minimum modulation detection threshold at 2 cycles/octave, and older listeners with normal hearing were remarkably similar to those of the young listeners. SMTFs for older listeners with mild and moderate hearing loss had a low-pass rather than a bandpass shape. Excitation patterns revealed that limited spectral resolution dictated modulation detection thresholds at high but not low spectral modulation frequencies. Even when factoring out (presumed) differences in frequency resolution among groups, the spectral envelope perception was worse for the group with moderate hearing loss than the other 3 groups. Conclusions The spectral envelope perception as measured by spectral modulation detection thresholds is compromised by hearing loss at higher spectral modulation frequencies, consistent with predictions of reduced spectral resolution known to accompany sensorineural hearing loss. Spectral envelope perception is not negatively impacted by advancing age at any spectral modulation frequency between 0.5 and 8.0 cycles/octave.

Purpose Human auditory nerve (AN) activity estimated from the amplitude of the first prominent negative peak (N1) of the compound action potential (CAP) is typically quantified using either a peak-to-peak measurement or a baseline-corrected measurement. However, the reliability of these 2 common measurement techniques has not been evaluated but is often assumed to be relatively poor, especially for older adults. To address this question, the current study (a) compared test–retest reliability of these 2 methods and (b) tested the extent to which measurement type affected the relationship between N1 amplitude and experimental factors related to the stimulus (higher and lower intensity levels) and participants (younger and older adults). Method Click-evoked CAPs were recorded in 24 younger (aged 18–30 years) and 20 older (aged 55–85 years) adults with clinically normal audiograms up to 3000 Hz. N1 peak amplitudes were estimated from peak-to-peak measurements (from N1 to P1) and baseline-corrected measurements for 2 stimulus levels (80 and 110 dB pSPL). Baseline-corrected measurements were made with 4 baseline windows. Each stimulus level was presented twice, and test–retest reliability of these 2 measures was assessed using the intraclass correlation coefficient. Linear mixed models were used to evaluate the extent to which age group and click level uniquely predicted N1 amplitude and whether the predictive relationships differed between N1 measurement techniques. Results Both peak-to-peak and baseline-corrected measurements of N1 amplitude were found to have good-to-excellent reliability, with intraclass correlation coefficient values > 0.60. As expected, N1 amplitudes were significantly larger for younger participants compared with older participants for both measurement types and were significantly larger in response to clicks presented at 110 dB pSPL than at 80 dB pSPL for both measurement types. Furthermore, the choice of baseline window had no significant effect on N1 amplitudes using the baseline-corrected method. Conclusions Our results suggest that measurements of AN activity can be robustly and reliably recorded in both younger and older adults using either peak-to-peak or baseline-corrected measurements of the N1 of the CAP. Peak-to-peak measurements yield larger N1 response amplitudes and are the default measurement type for many clinical systems, whereas baseline-corrected measurements are computationally simpler. Furthermore, the relationships between AN activity and stimulus- and participant-related variables were not affected by measurement technique, which suggests that these relationships can be compared across studies using different techniques for measuring the CAP N1.

Background: To confirm levels and detection timing of circulating microRNAs (miRNAs) in the serum of a mouse model for diagnosis of ototoxicity, circulating miR-205 in the serum was evaluated to reflect damages in the cochlear microstructure and compared to a kidney injury model. Method: A microarray for miRNAs in the serum was performed to assess the ototoxic effects of kanamycin-furosemide. Changes in the levels for the selected miRNAs (miR-205, miR-183, and miR-103) were compared in the serum and microstructures of the cochlea (stria vascularis, organ of Corti, and modiolus) between the ototoxicity and normal mouse groups. An acute kidney injury (AKI) mouse model was used to assess changes in miR-205 levels in the kidney by ototoxic drugs. Results: In the mouse model for ototoxicity, the serum levels of circulating miR-205 peaked on day 3 and were sustained from days 7–14. Furthermore, miR-205 expression was highly expressed in the organ of Corti at day 5, continued to be expressed in the modiolus at high levels until day 14, and was finally also in the stria vascularis. The serum miR-205 in the AKI mice did not change significantly compared to the normal group. Conclusions Circulating miR-205 from the cochlea, after ototoxic damage, migrates through the blood vessels to organs, which is then finally found in blood. In conditions of hearing impairment with ototoxic medications, detection of circulating miR-205 in the blood can be used to determine the extent of hearing loss. In the future, inner ear damage can be identified by simply performing a blood test before the hearing impairment due to ototoxic drugs.

Naked mole-rats are extremely social and extremely vocal rodents, displaying a wide range of functionally distinct call types and vocalizing almost continuously. Their vocalizations are low frequency, and a behavioral audiogram has shown that naked mole-rats, like other subterranean mammals, hear only low frequencies. Hence, the frequency range of their hearing and vocalizations appears to be well matched. However, even at low frequencies, naked mole-rats show very poor auditory thresholds, suggesting vocal communication may be effective only over short distances. However, in a tunnel environment where low frequency sounds propagate well and background noise is low, it may be that vocalizations travel considerable distances at suprathreshold intensities. Here, we confirmed hearing sensitivity using the auditory brainstem response; we characterized signature and alarm calls in intensity and frequency domains and we measured the effects of propagation through tubes with the diameter of naked mole-rat tunnels. Signature calls-used for intimate communication-could travel 3-8 m at suprathreshold intensities, and alarm calls (lower frequency and higher intensity), could travel up to 15 m. Despite this species' poor hearing sensitivity, the naked mole-rat displays a functional, coupled auditory-vocal communication system-a hallmark principle of acoustic communication systems across taxa.

Background: Numerous studies have demonstrated that individuals exhibit structured neural activity in many brain regions during rest that is also observed during different tasks, however it is still not clear whether and how resting state activity patterns may relate to underlying tuning for specific stimuli. In the posterior superior temporal gyrus (STG), distinct neural activity patterns are observed during the perception of specific linguistic speech features. We hypothesized that spontaneous resting-state neural dynamics of the STG would be structured to reflect its role in speech perception, exhibiting an organization along speech features as seen during speech perception. Methods: Human cortical local field potentials were recorded from the superior temporal gyrus (STG) in 8 patients undergoing surgical treatment of epilepsy. Signals were recorded during speech perception and rest. Patterns of neural activity (high gamma power: 70-150 Hz) during rest, extracted with spatiotemporal principal component analysis, were compared to spatiotemporal neural responses to speech features during perception. Hierarchical clustering was applied to look for patterns in rest that corresponded to speech feature tuning. Results: Significant correlations were found between neural responses to speech features (sentence onsets, consonants, and vowels) and the spontaneous neural activity in the STG. Across subjects, these correlations clustered into five groups, demonstrating tuning for speech features-most robustly for acoustic onsets. These correlations were not seen in other brain areas, or during motor and spectrally-rotated speech control tasks. Conclusions: In this study, we present evidence that the RS structure of STG activity robustly recapitulates its stimulus-evoked response to acoustic onsets. Further, secondary patterns in RS activity appear to correlate with stimulus-evoked responses to speech features. The role of these spontaneous spatiotemporal activity patterns remains to be elucidated.

To function as a mechanism in premating isolation, the divergent and species-specific calling songs of acoustic insects must be reliably processed by the afferent auditory pathway of receivers. Here, we analysed the responses of interneurons in a katydid species that uses long-lasting acoustic trills and compared these with previously reported data for homologous interneurons of a sympatric species that uses short chirps as acoustic signals. Some interneurons of the trilling species respond exclusively to the heterospecific chirp due to selective, low-frequency tuning and "novelty detection". These properties have been considered as evolutionary adaptations in the sensory system of the chirper, which allow it to detect signals effectively during the simultaneous calling of the sympatric sibling species. We propose that these two mechanisms, shared by the interneurons of both species, did not evolve in the chirper to guarantee its ability to detect the chirp under masking conditions. Instead we suggest that chirpers evolved an additional, 2-kHz component in their song and exploited pre-existing neuronal properties for detecting their song under masking noise. The failure of some interneurons to respond to the conspecific song in trillers does not prevent intraspecific communication, as other interneurons respond to the trill.

Auditory streaming is the process by which environmental sound is segregated into discrete perceptual objects. The auditory system has a remarkable capability in this regard as revealed in psychophysical experiments in humans and other primates. However, little is known about the underlying neuronal mechanisms, in part because of the lack of suitable behavioural paradigms in non-primate species. The mouse is an increasingly popular model for studying the neural mechanisms of perception and action because of the range of molecular tools enabling precise manipulation of neural circuitry. Here we present a novel behavioural task that can be used to assess perceptual aspects of auditory streaming in head-fixed mice. Animals were trained to detect a target sound in a one of two simultaneously presented, isochronous pure tone sequences. Temporal expectation was manipulated by presenting the target sound in a particular stream either early (~2 s) or late (~4 s) with respect to trial onset in blocks of 25-30 trials. Animals reached high performance on this task (d' > 1 overall), and notably their false alarms were very instructive of their behavioural state. Indeed, false alarm timing was markedly delayed for late blocks compared to early ones, indicating that the animals associated a different context to an otherwise identical stimulus. More finely, we observed that the false alarms were timed to the onset of the sounds present in the target stream. This suggests that the animals could selectively follow the target stream despite the presence of a distractor stream. Extracellular electrophysiological recordings during the task revealed that sound processing is flexibly modulated in a manner consistent with the optimisation of behavioural outcome. Together, these results indicate that the perceptual streaming can be inferred via the timing of false alarms in mice, and provide a new paradigm with which to investigate neuronal mechanisms of selective attention.

Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for kinocilium-oriented hair bundle function in cochlear hair cells.

Objective Age‐related muscle atrophy of the laryngeal muscles contributes to presbyphonia. Remodeling of the neuromuscular junction is one aspect underlying age‐related muscle atrophy. Although muscle disuse has been shown to exacerbate age‐related neuromuscular changes in the limb muscles, it is unknown if reduced vocal use has a similar effect in the laryngeal muscles. The objective of this study was to examine the use of social isolation as a novel method to reduce vocal use in old rats—and the impact of that reduced vocal use on ultrasonic vocalization acoustics and neuromuscular junction morphology in the thyroarytenoid muscle. Study Design Animal group comparison. Methods Old F344/BN rats (31 months of age) were socially isolated (n = 8) or communally housed (n = 8) for 8 weeks. Effect of housing condition on ultrasonic vocalization acoustics was assessed by calculating the changes in vocalization fundamental frequency and amplitude from baseline to 8 weeks. Neuromuscular junction morphology was measured in the lateral and medial portions of the thyroarytenoid muscle at the conclusion of the experiment. Results Vocalization amplitude decreased by a mean of −4.4 dB (standard deviation [SD], 4.49) after social isolation, whereas amplitude increased by a mean of 5.7 dB (SD, 5.07) in the communally housed rats ( P = 0.002). There was no significant difference in the change in fundamental frequency between groups. Furthermore, there were no group differences in any measure of neuromuscular junction morphology. Conclusion These results suggest that neuromuscular junctions in the thyroarytenoid muscle of old rats are unaffected by 8 weeks of social isolation, despite functional changes in vocalizations. Level of Evidence NA Laryngoscope , 129:E9–E14, 2019