Chronic stress induced loudness hyperacusis, sound avoidance and auditory cortex hyperactivity.

Hyperacusis, a debilitating loudness intolerance disorder, has been linked to chronic stress and adrenal insufficiency. To investigate the role of chronic stress, rats were chronically treated with corticosterone (CORT) stress hormone. Chronic CORT produced behavioral evidence of loudness hyperacusis, sound avoidance hyperacusis, and abnormal temporal integration of loudness. CORT treatment did not disrupt cochlear or brainstem function as reflected by normal distortion product otoacoustic emissions, compound action potentials, acoustic startle reflexex, and auditory brainstem responses. In contrast, the evoked response from the auditory cortex was enhanced up to three fold after CORT treatment. This hyperactivity was associated with a significant increase in glucocorticoid receptors in auditory cortex layers II/III and VI. Basal serum CORT levels remained normal after chronic CORT stress whereas reactive serum CORT levels evoked by acute restraint stress were blunted (reduced) after chronic CORT stress; similar changes were observed after chronic, intense noise stress. Taken together, our results show for the first time that chronic stress can induce hyperacusis and sound avoidance. A model is proposed in which chronic stress creates a subclinical state of adrenal insufficiency that establishes the necessary conditions for inducing hyperacusis.

Hyperacusis: Loudness intolerance, fear, annoyance and pain.

Hyperacusis is a debilitating loudness intolerance disorder that can evoke annoyance, fear and aural facial pain. Although the auditory system seems to be the "central" player, hyperacusis is linked to more than twenty non-auditory medical disorders such as Williams syndrome, autism spectrum disorder, fibromyalgia, migraine, head trauma, lupus and acoustic shock syndrome. Neural models suggest that some forms of hyperacusis may result from enhanced central gain, a process by which neural signals from a damaged cochlea are progressively amplified as activity ascends rostrally through the classical auditory pathway as well as other non-auditory regions of the brain involved in emotions, memory and stress. Imaging studies have begun to reveal the extended neural networks and patterns of functional connectivity in the brain that enrich sounds with negative attributes that can make listening unbearable and even painful. The development of animal models of hyperacusis have enabled researcher to begin to critically evaluate the biological bases of hyperacusis, identify therapies to ameliorate the symptoms and gain a better understanding of the neural mechanisms involved in loudness coding in normal and hearing impaired subjects.

A new mouse model of Charcot-Marie-Tooth 2J neuropathy replicates human axonopathy and suggest alteration in axo-glia communication.

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in MpzT124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along MpzT124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the MpzT124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

Galectin-3 protects auditory function in female mice.

Sex differences in the development of sensorineural hearing loss have been recognized in various inner ear disorders, but the molecular basis for such differences is poorly understood. Autosomal genes have been shown to cause sex differences in disease susceptibility, but many genes exerting sex-dependent effects on auditory function remain to be identified. Galectin-3 (Gal-3), a protein encoded by the autosomal gene Lgals3, is a member of the ß-galactoside-binding protein family, and has been linked to multiple biological processes, including immune responses, apoptosis, and cell adhesion. Here, we investigated auditory function and hair cell integrity in Gal-3 knockout (KO, Lgals3-/-) and wild-type (WT, Lgals3+/+) mice from age 1 to 6 months. KO mice show a more rapid age-related increase in ABR thresholds compared to WT mice. Noticeably, the threshold deterioration in female KO mice is significantly greater than in the male KO and WT mice. The ABR threshold elevation manifests over a broad frequency range in female KO mice, whereas the threshold elevations are confined to high frequencies in the male KO and WT mice. Moreover, DPOAE input/output functions reveal a similar pattern of auditory dysfunction, with the female KO mice displaying a significantly greater reduction in DPOAE amplitudes than male KO mice and WT mice of both sexes. Finally, age-related outer hair cell loss is greater for female KO mice compared to male KO mice and WT mice of both sexes. Together, these results indicate that Gal-3 deficiency exacerbates age-related cochlear degeneration and auditory dysfunction in female mice. Our study identifies Gal-3 as a sex-dependent molecule for maintaining female cochlear integrity.

Unexpected Consequences of Noise-Induced Hearing Loss: Impaired Hippocampal Neurogenesis, Memory, and Stress.

Noise-induced hearing loss (NIHL), caused by direct damage to the cochlea, reduces the flow of auditory information to the central nervous system, depriving higher order structures, such as the hippocampus with vital sensory information needed to carry out complex, higher order functions. Although the hippocampus lies outside the classical auditory pathway, it nevertheless receives acoustic information that influence its activity. Here we review recent results that illustrate how NIHL and other types of cochlear hearing loss disrupt hippocampal function. The hippocampus, which continues to generate new neurons (neurogenesis) in adulthood, plays an important role in spatial navigation, memory, and emotion. The hippocampus, which contains place cells that respond when a subject enters a specific location in the environment, integrates information from multiple sensory systems, including the auditory system, to develop cognitive spatial maps to aid in navigation. Acute exposure to intense noise disrupts the place-specific firing patterns of hippocampal neurons, "spatially disorienting" the cells for days. More traumatic sound exposures that result in permanent NIHL chronically suppresses cell proliferation and neurogenesis in the hippocampus; these structural changes are associated with long-term spatial memory deficits. Hippocampal neurons, which contain numerous glucocorticoid hormone receptors, are part of a complex feedback network connected to the hypothalamic-pituitary (HPA) axis. Chronic exposure to intense intermittent noise results in prolonged stress which can cause a persistent increase in corticosterone, a rodent stress hormone known to suppress neurogenesis. In contrast, a single intense noise exposure sufficient to cause permanent hearing loss produces only a transient increase in corticosterone hormone. Although basal corticosterone levels return to normal after the noise exposure, glucocorticoid receptors (GRs) in the hippocampus remain chronically elevated. Thus, NIHL disrupts negative feedback from the hippocampus to the HPA axis which regulates the release of corticosterone. Preclinical studies suggest that the noise-induced changes in hippocampal place cells, neurogenesis, spatial memory, and glucocorticoid receptors may be ameliorated by therapeutic interventions that reduce oxidative stress and inflammation. These experimental results may provide new insights on why hearing loss is a risk factor for cognitive decline and suggest methods for preventing this decline.

Blast trauma affects production and perception of mouse ultrasonic vocalizations.

Blast trauma from explosions affects hearing and communication in a significant proportion of soldiers. Many veterans report difficulty communicating, especially in noisy and reverberant environments, which contributes to complex mental health problems including anxiety and depression. However, the relationship between communication and perceptual problems after a blast has received little scientific attention. In the current studies, the effects of blast trauma on the production and perception of ultrasonic vocalizations (USVs) by CBA/CaJ mice, a common animal model for hearing and communication disorders, was explored. Overall, mice change the total number of vocalizations, the proportion produced of each syllable category, and the peak frequency, bandwidth, and duration of their vocalizations after blast exposure. Further, the perception of USVs is affected after blast trauma, with an immediate worsening of detection for most USV categories in the first 1-5 days after blasts, which later recovers. This study is the first to examine changes in the production and perception of communication signals after blast traumas in mice and is an important step towards developing treatments for blast-induced hearing and communication disorders.

Time- and frequency-dependent changes in acoustic startle reflex amplitude following cyclodextrin-induced outer and inner cell loss.

The acoustic startle reflex (ASR) amplitude can be enhanced or suppressed by noise-induced hearing loss or age-related hearing loss; however, little is known about how the ASR changes when ototoxic drugs destroy outer hair cells (OHCs) and inner hair cells (IHCs). High doses of 2-hydroxypropyl-beta-cyclodextrin (HPßCD), a cholesterol-lowering drug used to treat Niemann-Pick Type disease type C1, initially destroy OHCs and then the IHCs 6-8 weeks later. Adult rats were treated with doses of HPßCD designed to produce a diversity of hair cell lesions and hearing losses. When HPßCD destroyed OHCs and IHCs in the extreme base of the cochlea and caused minimal high-frequency hearing loss, the ASR amplitudes were enhanced at 4-, 8- and 16 kHz. Enhanced ASR occurred during the first few weeks post-treatment when only OHCs were missing; little change in the ASR occurred 6-8-WK post-treatment. If HPßCD destroyed most OHCs and many IHCs in the basal half of the cochlea, high-frequency thresholds increased ∼50 dB, and ASR amplitudes were reduced ∼50% at 4-, 8- and 16-kHz. The ASR amplitude reduction occurred in the first few weeks post-treatment when the OHCs were degenerating. The ASR was largely abolished when most of the OHCs were missing over the basal two-thirds of the cochlea and a 40-50 dB hearing loss was present at most frequencies. These results indicate that high-doses of HPßCD generally lead to a decline in ASR amplitude as OHCs degenerate; however, ASR amplitudes were enhanced in a few cases when hair cell loss was confined to the extreme base of the cochlea.

Combined antioxidants and anti-inflammatory therapies fail to attenuate the early and late phases of cyclodextrin-induced cochlear damage and hearing loss.

Niemann-Pick C1 (NPC1) is a fatal neurodegenerative disease caused by aberrant cholesterol metabolism. The progression of the disease can be slowed by removing excess cholesterol with high-doses of 2-hyroxypropyl-beta-cyclodextrin (HPßCD). Unfortunately, HPßCD causes hearing loss; the initial first phase involves a rapid destruction of outer hair cells (OHCs) while the second phase, occurring 4-6 weeks later, involves the destruction of inner hair cells (IHCs), pillar cells, collapse of the organ of Corti and spiral ganglion neuron degeneration. To determine whether the first and/or second phase of HPßCD-induced cochlear damage is linked, in part, to excess oxidative stress or neuroinflammation, rats were treated with a single-dose of 3000 mg/kg HPßCD alone or together with one of two combination therapies. Each combination therapy was administered from 2-days before to 6-weeks after the HPßCD treatment. Combination 1 consisted of minocycline, an antibiotic that suppresses neuroinflammation, and HK-2, a multifunctional redox modulator that suppresses oxidative stress. Combination 2 was comprised of minocycline plus N-acetyl cysteine (NAC), which upregulates glutathione, a potent antioxidant. To determine if either combination therapy could prevent HPßCD-induced hearing impairment and cochlear damage, distortion product otoacoustic emissions (DPOAE) were measured to assess OHC function and the cochlear compound action potential (CAP) was measured to assess the function of IHCs and auditory nerve fibers. Cochleograms were prepared to quantify the amount of OHC, IHC and pillar cell (PC) loss. HPßCD significantly reduced DPOAE and CAP amplitudes and caused significant OHC, IHC and OPC losses with losses greater in the high-frequency base of the cochlea than the apex. Neither minocycline + HK-2 (MIN+ HK-2) nor minocycline + NAC (MIN+NAC) prevented the loss of DPOAEs, CAPs, OHCs, IHCs or IPCs caused by HPßCD. These results suggest that oxidative stress and neuroinflammation are unlikely to play major roles in mediating the first or second phase of HPßCD-induced cochlear damage. Thus, HPßCD-induced ototoxicity must be mediated by some other unknown cell-death pathway possibly involving loss of trophic support from damaged support cells or disrupted cholesterol metabolism.

Auditory hypersensitivity and processing deficits in a rat model of fragile X syndrome.

Fragile X (FX) syndrome is one of the leading inherited causes of autism spectrum disorder (ASD). A majority of FX and ASD patients exhibit sensory hypersensitivity, including auditory hypersensitivity or hyperacusis, a condition in which everyday sounds are perceived as much louder than normal. Auditory processing deficits in FX and ASD also afford the opportunity to develop objective and quantifiable outcome measures that are likely to translate between humans and animal models due to the well-conserved nature of the auditory system and well-developed behavioral read-outs of sound perception. Therefore, in this study we characterized auditory hypersensitivity in a Fmr1 knockout (KO) transgenic rat model of FX using an operant conditioning task to assess sound detection thresholds and suprathreshold auditory reaction time-intensity (RT-I) functions, a reliable psychoacoustic measure of loudness growth, at a variety of stimulus frequencies, bandwidths, and durations. Male Fmr1 KO and littermate WT rats both learned the task at the same rate and exhibited normal hearing thresholds. However, Fmr1 KO rats had faster auditory RTs over a broad range of intensities and steeper RT-I slopes than WT controls, perceptual evidence of excessive loudness growth in Fmr1 KO rats. Furthermore, we found that Fmr1 KO animals exhibited abnormal perceptual integration of sound duration and bandwidth, with diminished temporal but enhanced spectral integration of sound intensity. Because temporal and spectral integration of sound stimuli were altered in opposite directions in Fmr1 KO rats, this suggests that abnormal RTs in these animals are evidence of aberrant auditory processing rather than generalized hyperactivity or altered motor responses. Together, these results are indicative of fundamental changes to low-level auditory processing in Fmr1 KO animals. Finally, we demonstrated that antagonism of metabotropic glutamate receptor 5 (mGlu5) selectively and dose-dependently restored normal loudness growth in Fmr1 KO rats, suggesting a pharmacologic approach for alleviating sensory hypersensitivity associated with FX. This study leverages the tractable nature of the auditory system and the unique behavioral advantages of rats to provide important insights into the nature of a centrally important yet understudied aspect of FX and ASD.

Loss of CX3CR1 augments neutrophil infiltration into cochlear tissues after acoustic overstimulation.

The cochlea, the sensory organ for hearing, has a protected immune environment, segregated from the systemic immune system by the blood-labyrinth barrier. Previous studies have revealed that acute acoustic injury causes the infiltration of circulating leukocytes into the cochlea. However, the molecular mechanisms controlling immune cell trafficking are poorly understood. Here, we report the role of CX3CR1 in regulating the entry of neutrophils into the cochlea after acoustic trauma. We employed B6.129P-Cx3cr1tm1Litt /J mice, a transgenic strain that lacks the gene, Cx3cr1, for coding the fractalkine receptor. Our results demonstrate that lack of Cx3cr1 results in the augmentation of neutrophil infiltration into cochlear tissues after exposure to an intense noise of 120 dB SPL for 1 hr. Neutrophil distribution in the cochlea is site specific, and the infiltration level is positively associated with noise intensity. Moreover, neutrophils are short lived and macrophage phagocytosis plays a role in neutrophil clearance, consistent with typical neutrophil dynamics in inflamed non-cochlear tissues. Importantly, our study reveals the potentiation of noise-induced hearing loss and sensory cell loss in Cx3cr1-/- mice. In wild-type control mice (Cx3cr1+/+ ) exposed to the same noise, we also found neutrophils. However, neutrophils were present primarily inside the microvessels of the cochlea, with only a few in the cochlear tissues. Collectively, our data implicate CX3CR1-mediated signaling in controlling neutrophil migration from the circulation into cochlear tissues and provide a better understanding of the impacts of neutrophils on cochlear responses to acoustic injury.

Spatiotemporal Developmental Upregulation of Prestin Correlates With the Severity and Location of Cyclodextrin-Induced Outer Hair Cell Loss and Hearing Loss.

2-Hyroxypropyl-beta-cyclodextrin (HPßCD) is being used to treat Niemann-Pick C1, a fatal neurodegenerative disease caused by abnormal cholesterol metabolism. HPßCD slows disease progression, but unfortunately causes severe, rapid onset hearing loss by destroying the outer hair cells (OHC). HPßCD-induced damage is believed to be related to the expression of prestin in OHCs. Because prestin is postnatally upregulated from the cochlear base toward the apex, we hypothesized that HPßCD ototoxicity would spread from the high-frequency base toward the low-frequency apex of the cochlea. Consistent with this hypothesis, cochlear hearing impairments and OHC loss rapidly spread from the high-frequency base toward the low-frequency apex of the cochlea when HPßCD administration shifted from postnatal day 3 (P3) to P28. HPßCD-induced histopathologies were initially confined to the OHCs, but between 4- and 6-weeks post-treatment, there was an unexpected, rapid and massive expansion of the lesion to include most inner hair cells (IHC), pillar cells (PC), peripheral auditory nerve fibers, and spiral ganglion neurons at location where OHCs were missing. The magnitude and spatial extent of HPßCD-induced OHC death was tightly correlated with the postnatal day when HPßCD was administered which coincided with the spatiotemporal upregulation of prestin in OHCs. A second, massive wave of degeneration involving IHCs, PC, auditory nerve fibers and spiral ganglion neurons abruptly emerged 4-6 weeks post-HPßCD treatment. This secondary wave of degeneration combined with the initial OHC loss results in a profound, irreversible hearing loss.

Roles of Bak and Sirt3 in Paraquat-Induced Cochlear Hair Cell Damage.

Paraquat, a superoxide generator, can damage the cochlea causing an ototoxic hearing loss. The purpose of the study was to determine if deletion of Bak, a pro-apoptotic gene, would reduce paraquat ototoxicity or if deletion of Sirt3, which delays age-related hearing loss under caloric restriction, would increase paraquat ototoxicity. We tested these two hypotheses by treating postnatal day 3 cochlear cultures from Bak±, Bak-/-, Sirt3±, Sirt3-/-, and WT mice with paraquat and compared the results to a standard rat model of paraquat ototoxicity. Paraquat damaged nerve fibers and dose-dependently destroyed rat outer hair cells (OHCs) and inner hair cells (IHCs). Rat hair cell loss began in the base of the cochlea with a 10 µM dose and as the dose increased from 50 to 500 µM, the hair cell loss increased near the base of the cochlea and spread toward the apex of the cochlea. Rat OHC losses were consistently greater than IHC losses. Unexpectedly, in all mouse genotypes, paraquat-induced hair cell lesions were maximal near the apex of the cochlea and minimal near the base. This unusual damage gradient is opposite to that seen in paraquat-treated rats and in mice and rats treated with other ototoxic drugs. However, paraquat always induced greater OHC loss than IHC loss in all mouse strains. Contrary to our hypothesis, Bak deficient mice were more vulnerable to paraquat ototoxicity than WT mice (Bak-/- > Bak± > WT), suggesting that Bak plays a protective role against hair cell stress. Also, contrary to expectation, Sirt3-deficient mice did not differ significantly from WT mice, possibly due to the fact that Sirt3 was not experimentally upregulated in Sirt3-expressing mice prior to paraquat treatment. Our results show for the first time a gradient of ototoxic damage in mice that is greater in the apex than the base of the cochlea.

Long term changes to auditory sensitivity following blast trauma in mice.

Blast trauma is a common acoustic/physical insult occurring in modern warfare. Twenty percent of active duty military come into close proximity to explosions and experience mild to severe sensory deficits. The prevalence of such injuries is high but correlating auditory sensitivity changes with the initial insult is difficult because injury and evaluations are often separated by long time periods. Here, auditory sensitivity was measured before and after a traumatic blast in adult CBA/CaJ mice using auditory brainstem responses, distortion production otoacoustic emissions, and behavioral detection of pure tones. These measurements included baseline auditory sensitivity prior to injury in all mice, and again at 3, 30, and 90 days after the blast in the two physiological groups, and daily for up to 90 days in the behavioral group. Mice in all groups experienced an initial deterioration in auditory sensitivity, though physiological measurements showed evidence of recovery that behavioral measurements did not. Amplitudes and latencies of ABR waves may reflect additional changes beyond the peripheral damage shown by the threshold changes and should be explored further. The present work addresses a major gap in the current acoustic trauma literature both in terms of comparing physiological and behavioral methods, as well as measuring the time course of recovery.

Review: Neural Mechanisms of Tinnitus and Hyperacusis in Acute Drug-Induced Ototoxicity.

Purpose Tinnitus and hyperacusis are debilitating conditions often associated with age-, noise-, and drug-induced hearing loss. Because of their subjective nature, the neural mechanisms that give rise to tinnitus and hyperacusis are poorly understood. Over the past few decades, considerable progress has been made in deciphering the biological bases for these disorders using animal models. Method Important advances in understanding the biological bases of tinnitus and hyperacusis have come from studies in which tinnitus and hyperacusis are consistently induced with a high dose of salicylate, the active ingredient in aspirin. Results Salicylate induced a transient hearing loss characterized by a reduction in otoacoustic emissions, a moderate cochlear threshold shift, and a large reduction in the neural output of the cochlea. As the weak cochlear neural signals were relayed up the auditory pathway, they were progressively amplified so that the suprathreshold neural responses in the auditory cortex were much larger than normal. Excessive central gain (neural amplification), presumably resulting from diminished inhibition, is believed to contribute to hyperacusis and tinnitus. Salicylate also increased corticosterone stress hormone levels. Functional imaging studies indicated that salicylate increased spontaneous activity and enhanced functional connectivity between structures in the central auditory pathway and regions of the brain associated with arousal (reticular formation), emotion (amygdala), memory/spatial navigation (hippocampus), motor planning (cerebellum), and motor control (caudate/putamen). Conclusion These results suggest that tinnitus and hyperacusis arise from aberrant neural signaling in a complex neural network that includes both auditory and nonauditory structures.

Functional Neuroanatomy of Salicylate- and Noise-Induced Tinnitus and Hyperacusis.

Tinnitus and hyperacusis are debilitating conditions often associated with aging or exposure to intense noise or ototoxic drugs. One of the most reliable methods of inducing tinnitus is with high doses of sodium salicylate, the active ingredient in aspirin. High doses of salicylate have been widely used to investigate the functional neuroanatomy of tinnitus and hyperacusis. High doses of salicylate have been used to develop novel behavioral methods to detect the presence of tinnitus and hyperacusis in animal models. Salicylate typically induces a hearing loss of approximately 20 dB which greatly reduces the neural output of the cochlea. As this weak neural signal emerging from the cochlea is sequentially relayed to the cochlear nucleus, inferior colliculus, medial geniculate, and auditory cortex, the neural response to suprathreshold sounds is progressively amplified by a factor of 2-3 by the time the signal reaches the auditory cortex, a phenomenon referred to as enhanced central gain. Sound-evoked hyperactivity also occurred in the amygdala, a region that assigns emotional significance to sensory stimuli. Resting state functional magnetic imaging of the BOLD signal revealed salicylate-induced increases in spontaneous neural activity in the inferior colliculus, medial geniculate body, and auditory cortex as well as in non-auditory areas such as the amygdala, reticular formation, cerebellum, and other sensory areas. Functional connectivity of the BOLD signal revealed increased neural coupling between several auditory areas and non-auditory areas such as the amygdala, cerebellum, reticular formation, hippocampus, and caudate/putamen; these strengthened connections likely contribute to the multifaceted dimensions of tinnitus. Taken together, these results suggest that salicylate-induced tinnitus disrupts a complex neural network involving many auditory centers as well as brain regions involved with emotion, arousal, memory, and motor planning. These extra-auditory centers embellish the basic auditory percepts that results in tinnitus and which may also contribute to hyperacusis.

Hydroxypropyl-ß-cyclodextrin causes massive damage to the developing auditory and vestibular system.

2-hydroxypropyl-ß-cyclodextrin (HPßCD), a cholesterol chelator used to treat Niemann-Pick C1 (NPC1) lysosomal storage disease, causes hearing loss in mammals by preferentially destroying outer hair cells. Because cholesterol plays an important role in early neural development, we hypothesized that HPßCD would cause more extensive damage to postnatal cochlear and vestibular structures in than adult rats. This hypothesis was tested by administering HPßCD to adult rats and postnatal day 3 (P3) cochlear and vestibular organ cultures. Adult rats treated with HPßCD developed hearing impairment and outer hair cell loss 3-day post-treatment; damage increased with dose from the high frequency base toward the low-frequency apex. The HPßCD-induced histopathologies were more severe and widespread in cochlear and vestibular cultures at P3 than in adults. HPßCD destroyed both outer and inner hair cells, auditory nerve fibers and spiral ganglion neurons as well as type I and type II vestibular hair cells and vestibular ganglion neurons. The early stage of HPßCD damage involved disruption of hair cell mechanotransduction and destruction of stereocilia. HPßCD-mediated apoptosis in P3 cultures was most-strongly initiated by activation of the extrinsic caspase-8 cell death pathway in cochlear and vestibular hair cells and neurons followed by activation of executioner caspase-3. Thus, HPßCD is toxic to all types of postnatal cochlear and vestibular hair cells and neurons in vitro whereas in vivo it only appears to destroy outer hair cells in adult cochleae. The more severe HPßCD-induced damage in postnatal cultures could be due to greater drug bioavailability in vitro and/or greater vulnerability of the developing inner ear.

Blast-induced hearing loss suppresses hippocampal neurogenesis and disrupts long term spatial memory.

Acoustic information transduced by cochlear hair cells is continuously relayed from the auditory pathway to other sensory, motor, emotional and cognitive centers in the central nervous system. Human epidemiological studies have suggested that hearing loss is a risk factor for dementia and cognitive decline, but the mechanisms contributing to these memory and cognitive impairments are poorly understood. To explore these issues in a controlled experimental setting, we exposed adult rats to a series of intense blast wave exposures that significantly reduced the neural output of the cochlea. Several weeks later, we used the Morris Water Maze test, a hippocampal-dependent memory task, to assess the ability of Blast Wave and Control rats to learn a spatial navigation task (memory acquisition) and to remember what they had learned (spatial memory retention) several weeks earlier. The elevated plus maze and open field arena were used to test for anxiety-like behaviors. Afterwards, hippocampal cell proliferation and neurogenesis were evaluated using bromodeoxyuridine (BrdU), doublecortin (DCX), and Neuronal Nuclei (NeuN) immunolabeling. The Blast Wave and Control rats learned the spatial navigation task equally well and showed no differences on tests of anxiety. However, the Blast Wave rats performed significantly worse on the spatial memory retention task, i.e., remembering where they had been two weeks earlier. Deficits on the spatial memory retention task were associated with significant decreases in hippocampal cell proliferation and neurogenesis. Our blast wave results are consistent with other experimental manipulations that link spatial memory retention deficits (long term memory) with decreased cell proliferation and neurogenesis in the hippocampus. These results add to the growing body of knowledge linking blast-induced cochlear hearing loss with the cognitive deficits often seen in combat personnel and provide mechanistic insights into these extra auditory disorders that could lead to therapeutic interventions.

Interaction of auditory and pain pathways: Effects of stimulus intensity, hearing loss and opioid signaling.

Moderate intensity sounds can reduce pain sensitivity (i.e., audio-analgesia) whereas intense sounds can induce aural pain, evidence of multisensory interaction between auditory and pain pathways. To explore auditory-pain pathway interactions, we used the tail-flick (TF) test to assess thermal tail-pain sensitivity by measuring the latency of a rat to remove its tail from 52 °C water. In Experiment 1, TF latencies were measured in ambient noise and broadband noise (BBN) presented from 80 to 120 dB SPL. TF latencies gradually increased from ambient to 90 dB SPL (audio-analgesia), but then declined. At 120 dB, TF latencies were significantly shorter than normal, evidence for audio-hyperalgesia near the aural threshold for pain. In Experiment II, the opioid pain pathway was modified by treating rats with a high dose of fentanyl known to induce post-treatment hyperalgesia. TF latencies in ambient noise were normal 10-days post-fentanyl. However, TF latencies became shorter than normal from 90 to 110 dB indicating that fentanyl pre-treatment had converted audio-analgesia to audio-hyperalgesia. In Experiment III, we tested the hypothesis that hearing loss could alter pain sensitivity by unilaterally exposing rats to an intense noise that induced a significant hearing loss. TF latencies in ambient noise gradually declined from 1- to 4-weeks post-exposure indicating that noise-induced hearing loss had increased pain sensitivity. Our results suggest that auditory and pain pathways interact in ways that depend on intensity, hearing loss and opioid pain signaling, results potentially relevant to pain hyperacusis.

New insights on repeated acoustic injury: Augmentation of cochlear susceptibility and inflammatory reaction resultant of prior acoustic injury.

In industrial and military settings, individuals who suffer from one episode of acoustic trauma are likely to sustain another episode of acoustic stress, creating an opportunity for a potential interaction between the two stress conditions. We previously demonstrated that acoustic overstimulation perturbs the cochlear immune environment. However, how the cochlear immune system responds to repeated acoustic overstimulation is unknown. Here, we used a mouse model to investigate the cochlear immune response to repeated stress. We reveal that exposure to an intense noise at 120 dB SPL for 1 h activates the cochlear immune response in a time-dependent fashion with substantial expansion and activation of the macrophage population in the cochlea at 2-days post-exposure. At 20-days post-exposure, the number and pro-inflammatory phenotypes of cochlear macrophages have significantly subsided, but have yet to return to homeostatic levels. Monocytes with anti-inflammatory phenotypes are recruited into the cochlea. With the presence of this residual immune activation, a second exposure to the same noise provokes an exaggerated inflammatory response as evidenced by exacerbated maturation of macrophages. Furthermore, the second noise causes greater sensory cell pathogenesis. Unlike the first noise-induced damage that occurs mainly between 0 and 2 days post-exposure, the second noise-induced damage occurs more frequently between 2 and 20 days post-exposure, the period when secondary damage takes place. These observations suggest that repeated acoustic overstimulation exacerbates cochlear inflammation and secondary sensory cell pathogenesis. Together, our results suggest that the cochlear immune system plays an important role in modulating cochlear responses to repeated acoustic stress.

Dynamic Changes in Synaptic Plasticity Genes in Ipsilateral and Contralateral Inferior Colliculus Following Unilateral Noise-induced Hearing Loss.

Unilateral noise-induced hearing loss reduces the input to the central auditory pathway disrupting the excitatory and inhibitory inputs to the inferior colliculus (IC), an important binaural processing center. Little is known about the compensatory synaptic changes that occur in the IC as a consequence of unilateral noise-induced hearing loss. To address this issue, Sprague-Dawley rats underwent unilateral noise exposure resulting in severe unilateral hearing loss. IC tissues from the contralateral and ipsilateral IC were evaluated for acute (2-d) and chronic (28-d) changes in the expression of 84 synaptic plasticity genes on a PCR array. Arc and Egr1 genes were further visualized by in situ hybridization to validate the PCR results. None of the genes were upregulated, but many were downregulated post-exposure. At 2-d post-exposure, more than 75% of the genes were significantly downregulated in the contralateral IC, while only two were downregulated in the ipsilateral IC. Many of the downregulated genes were related to long-term depression, long-term potentiation, cell adhesion, immediate early genes, neural receptors and postsynaptic density. At 28-d post-exposure, the gene expression pattern was reversed with more than 85% of genes in the ipsilateral IC now downregulated. Most genes previously downregulated in the contralateral IC 2-d post-exposure had recovered; less than 15% remained downregulated. These time-dependent, asymmetric changes in synaptic plasticity gene expression could shed new light on the perceptual deficits associated with unilateral hearing loss and the dynamic structural and functional changes that occur in the IC days and months following unilateral noise-induced hearing loss.