Publications using the System 3

Platinum-based agents, such as cisplatin, form the mainstay of currently used chemotherapeutic regimens for several malignancies; however, the main limitations are chemoresistance and ototoxic side effects. In this study we used two different polyphenols, curcumin and ferulic acid as adjuvant chemotherapeutics evaluating (1) in vivo their antioxidant effects in protecting against cisplatin ototoxicity and (2) in vitro the transcription factors involved in tumor progression and cisplatin resistance. We reported that both polyphenols show antioxidant and oto-protective activity in the cochlea by up-regulating Nrf-2/HO-1 pathway and downregulating p53 phosphorylation. However, only curcumin is able to influence inflammatory pathways counteracting NF-κB activation. In human cancer cells, curcumin converts the anti-oxidant effect into a pro-oxidant and anti-inflammatory one. Curcumin exerts permissive and chemosensitive properties by targeting the cisplatin chemoresistant factors Nrf-2, NF-κB and STAT-3 phosphorylation. Ferulic acid shows a biphasic response: it is pro-oxidant at lower concentrations and anti-oxidant at higher concentrations promoting chemoresistance. Thus, polyphenols, mainly curcumin, targeting ROS-modulated pathways may be a promising tool for cancer therapy. Thanks to their biphasic activity of antioxidant in normal cells undergoing stressful conditions and pro-oxidant in cancer cells, these polyphenols probably engage an interplay among the key factors Nrf-2, NF-κB, STAT-3 and p53.

Parvalbumin-positive (PV+) interneurons are major regulators of adult experience-dependent plasticity. Acute manipulation of PV+ cell activity before learning alters the rate of acquisition of new skills, whereas transient inactivation of PV+ cells interferes with retrieval of previously learned information. However, the effects of sustained PV+ cell manipulation throughout training remain largely unknown. Using chemogenetics in rat auditory cortex during an adaptive sound disrimination task, here we show that PV+ cells exert bidirectional control over the rate of perceptual learning. Down-regulation of PV+ cell activity accelerated learning, but increasing their activity resulted in slower learning. However, both interventions led to reduced gains in perceptual acuity by the end of training relative to controls. Furthermore, longitudinal training performance was functionally correlated with measures of neural synchrony and stimulus-specific adaptation. These findings suggest that, whereas restricting PV+ cell activity may initially facilitate training-induced plasticity, a subsequent increase in PV+ cell activity is necessary to prevent further plastic changes and consolidate learning.

The formation of reactive oxygen species (ROS) is a well-documented process in noise-induced hearing loss (NIHL). We have also previously shown that activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPKα) at its catalytic residue T172 is one of the key reactions triggering noise-induced outer hair cell (OHC) death. In this study, we are addressing the link between ROS formation and activation of AMPKα in OHCs after noise exposure. In-vivo treatment of CBA/J mice with the antioxidant N-acetyl cysteine (NAC) reduced noise-induced ROS formation (as assessed by the relative levels of 4-hydroxynonenal and 3-nitrotyrosine) and activation of AMPKα in OHCs. Forskolin, an activator of adenylyl cyclase (AC) and an antioxidant, significantly increased cyclic adenosine monophosphate (cAMP) and decreased ROS formation and noise-induced activation of AMPKα. Consequently, treatment with forskolin attenuated noise-induced losses of OHCs and NIHL. In HEI-OC1 cells, H2O2-induced activation of AMPKα and cell death were inhibited by the application of forskolin. The sum of our data indicates that noise activates AMPKα in OHCs through formation of ROS and that noise-exposure-induced OHC death is mediated by a ROS/AMPKα-dependent pathway. Forskolin may serve as a potential compound for prevention of NIHL.

Background: Mitochondrial dysfunction is considered to contribute to the development of age-related hearing loss (AHL). The regulation of mitochondrial function requires mitochondrial quality control, which includes mitophagy and dynamics. Dynamin-related Protein 1 (DRP-1) is believed to play a central role in this regulation. However, the underlying mechanism of DRP-1 in AHL remains unclear. Here, we examined whether the decline of DRP-1-dependent mitophagy contributes to the development of AHL. Methods: We induced cellular and cochlear senescence using hydrogen peroxide (H2O2) and evaluated the level of senescence through senescence-associated β-galactosidase staining. We evaluated mitophagy levels via fluorescence imaging and Western Blotting of LC3II and P62. Mitochondrial function was assessed by ATP assay, mtDNA assay, and JC-1. Results: We found that both the expression of DRP-1 and the mitophagy level decreased in senescent cells and aged mice. DRP-1 overexpression in HEI-OC1 cells initiated mitophagy and preserved mitochondrial function when exposed to H2O2, while cells with DRP-1 silencing displayed otherwise. Moreover, inhibition of DRP-1 by Mdivi-1 blocked mitophagy and exacerbated hearing loss in aged C57BL/6 mice. Conclusion: These results indicated that DRP-1 initiated mitophagy, eliminated mitochondrial dysfunction, and may protect against oxidative stress-induced senescence. These results provide a potential therapeutic target for AHL.

The transient receptor potential cation channel, vanilloid type (TRPV) 3, is a member of the TRPV subfamily that is expressed predominantly in the skin, hair follicles, and gastrointestinal tract. It is also distributed in the organ of Corti of the inner ear and colocalizes with TRPV1 or TRPV4, but its role in auditory function is unknown. In the present study, we demonstrate that TRPV3 is expressed in inner hair cells (HCs) but mainly in cochlear outer HCs in mice, with expression limited to the cytoplasm and not detected in stereocilia. We compared the number of HCs as well as distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) thresholds between TRPV3 knockout (V3KO) and wild-type (V3WT) mice and found that although most mutants (72.3%) had normal hearing, a significant proportion (27.7%) showed impaired hearing associated with loss of cochlear HCs. Compensatory upregulation of TRPV4 in HCs prevented HC damage and kanamycin-induced hearing loss and preserved normal auditory function in most of these mice. Thus, TRPV4 and TRPV3 in cochlear HCs protect hearing in mice; moreover, the results suggest some functional redundancy in the functions of TRPV family members. Our findings provide novel insight into the molecular basis of auditory function in mammals that can be applied to the development of strategies to mitigate hearing loss.

The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporation-mediated transuterine gene transfer of Connexin30 , utilizing a monophasic pulse into Connexin30 −/− mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. However, it is not clear whether supplementary gene transfer can rescue significant morphological changes, caused by genetic deficits. In addition, with the transuterine gene transfer technique utilized in our previous report, the survival rate of embryos and their mothers after treatment was low, which became a serious obstacle for effective in vivo experiments. Here, we set out to elucidate the feasibility of supplementation therapy in Slc26a4 deficient mice, utilizing biphasic pulses, optimized by modifying pulse conditions. Modification of the biphasic pulse conditions during electroporation increased the survival rate. In addition, supplementation of the target gene cDNA into the otocysts of homozygous Slc24a4 knockout mice significantly prevented enlargement of the endolymphatic space in the inner ear areas; moreover, it rescued hearing and vestibular function of mice in vivo .

Objective Dietary intervention is a practical prevention strategy for age-related hearing loss (AHL). Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) may be effective in prevention of AHL due to their anti-inflammatory and tissue-protective functions. Age-related changes in the hearing function of wild-type and Fat - 1 transgenic mice derived from the C57BL/6N strain, which can convert omega-6 PUFAs to n-3 PUFAs and consequently produce enriched endogenous n-3 PUFAs, were investigated to test the efficacy of n-3 PUFAs for AHL prevention. Results At 2 months, the baseline auditory brainstem response (ABR) thresholds were the same in Fat - 1 and wild-type mice at 8–16 kHz but were significantly higher in Fat - 1 mice at 4 and 32 kHz. In contrast, the ABR thresholds of Fat - 1 mice were significantly lower at 10 months. Moreover, the ABR thresholds of Fat - 1 mice at low-middle frequencies were significantly lower at 13 months (12 kHz). Body weights were significantly reduced in Fat - 1 mice at 13 months, but not at 2, 10, and 16–17 months. In conclusion, enriched endogenous n-3 PUFAs produced due to the expression of the Fat - 1 transgene partially alleviated AHL in male C57BL/6N mice.

In auditory-conditioned fear learning, the freezing response is independent of the sound frequencies used, but the frequency of the conditioned sound is considered distinct from those of unrelated sounds based on electrophysiological responses in the auditory system. Whether an emergent discriminative learning underlies auditory fear conditioning and which nuclei and pathways are involved in it remain unclear. Using behavioral and electrophysiological assays, we found that the response of medial prefrontal cortex (mPFC) neurons to a conditioned auditory stimulus (CS) was enhanced relative to the response to unrelated frequencies (UFs) after auditory fear conditioning, and mice could distinguish the CS during multifrequency testing, a phenomenon called emergent discriminative learning. After silencing the mPFC with muscimol, emergent discriminative learning was blocked. In addition, the pure tone responses of mPFC neurons were inhibited after injection of lidocaine in the ipsilateral primary auditory cortex (A1), and the emergent discriminative learning was blocked by silencing both sides of A1 with muscimol. This study, therefore, provides evidence for an emergent discriminative learning mediated by mPFC and A1 neurons after auditory fear conditioning.

The ability to recognize sounds in noise is a key part of hearing, and the mechanisms by which the brain identifies sounds in noise are of considerable interest to scientists, clinicians and engineers. Yet we know little about the necessity of regions such as auditory cortex for hearing in noise, or how cortical processing of sounds is adversely affected by noise. Here we used reversible cortical inactivation and extracellular electrophysiology in ferrets performing a vowel discrimination task to identify and understand the causal contribution of auditory cortex to hearing in noise. Cortical inactivation by cooling impaired task performance in noisy but not clean conditions, while responses of auditory cortical neurons were less informative about vowel identity in noise. Simulations mimicking cortical inactivation indicated that effects of inactivation were related to the loss of information about sounds represented across neural populations. The addition of noise to target sounds drove spiking activity in auditory cortex and recruitment of additional neural populations that were linked to degraded behavioral performance. To suppress noise-related activity, we used continuous exposure to background noise to adapt the auditory system and recover behavioral performance in both ferrets and humans. Inactivation by cooling revealed that the benefits of continuous exposure were not cortically dependent. Together our results highlight the importance of auditory cortex in sound discrimination in noise and the underlying mechanisms through which noise-related activity and adaptation shape hearing.

The role of the claustrum in consciousness and vigilance states was proposed more than two decades ago; however, its role in anesthesia is not yet understood, and this requires more investigation. The aim of our study was to assess the impact of claustrum electrical stimulation during isoflurane anesthesia in adult rats. The claustrum in the left hemisphere was electrically stimulated using a bipolar tungsten electrode inserted stereotaxically. In order to monitor the anesthetic depth, the electrocorticogram (ECoG) was recorded before, during, and after claustrum stimulation using frontal and parietal epidural electrodes placed over the left hemisphere. After reaching stabilized slow-wave isoflurane anesthesia, twenty stimuli, each of one second duration with ten seconds interstimulus duration, were applied. ECoG analysis has shown that, after a delay from the beginning of stimulation, the slow-wave ECoG signal changed to a transient burst suppression (BS) pattern. Our results show that electrical stimulation of the claustrum area during slow-wave isoflurane anesthesia induces a transitory increase in anesthetic depth, documented by the appearance of a BS ECoG pattern, and suggests a potential role of claustrum in anesthesia.

Long noncoding RNA (lncRNA) disorder has been found in many kinds of age-associated diseases. However, the role of lncRNA in the development of age-related hearing loss (AHL) is still largely unknown. This study sought to uncover AHL-associated lncRNAs and the function. RNA-sequencing was conducted to profile lncRNA expression in the cochlea of an early-onset AHL mouse model. RT-qPCR assay was used to validate the expression pattern of lncRNAs. ATP assay, JC-1 assay, mitochondrial probe staining, CCK-8 assay, Western blot, and immunocytochemistry were performed to detect the effects of lncRNA AW112010 in HEI-OC1 cells and the mouse cochlea. We identified 88 significantly upregulated lncRNAs and 46 significantly downregulated lncRNAs in the cochlea of aged C57BL/6 mice. We focused on the significantly upregulated AW112010. Silencing of AW112010 decreased the ATP level, mitochondrial membrane potential, and cell viability and increased mitochondrial ROS generation under oxidative stress in HEI-OC1 cells. AW112010 overexpression promoted cell survival in HEI-OC1 cells. AW112010 knockdown reduced mitochondrial mass and impaired mitochondrial biogenesis in HEI-OC1 cells. Activation of mitochondrial biogenesis by resveratrol and STR1720 promoted cell survival. The mitochondrial biogenesis process was activated in the cochlea of aged mice. Moreover, AW112010 regulated AMPK signaling in HEI-OC1 cells. Transcription factor Arid5b elevated in the aged cochlea and induced AW112010 expression and mitochondrial biogenesis in HEI-OC1 cells. Taken together, lncRNAs are dysregulated with aging in the cochlea of C57BL/6 mice. The Arid5b/AW112010 signaling was induced in the aged mouse cochlea and positively modulated the mitochondrial biogenesis to maintain mitochondrial function.

Noise exposure affects the organ of Corti and the lateral wall of the cochlea, including the stria vascularis and spiral ligament. Although the inner ear vasculature and spiral ligament fibrocytes in the lateral wall consist of a significant proportion of cells in the cochlea, relatively little is known regarding their functional significance. In this study, 6-week-old male C57BL/6 mice were exposed to noise trauma to induce transient hearing threshold shift (TTS) or permanent hearing threshold shift (PTS). Compared to mice with TTS, mice with PTS exhibited lower cochlear blood flow and lower vessel diameter in the stria vascularis, accompanied by reduced expression levels of genes involved in vasodilation and increased expression levels of genes related to vasoconstriction. Ultrastructural analyses by transmission electron microscopy revealed that the stria vascularis and spiral ligament fibrocytes were more damaged by PTS than by TTS. Moreover, mice with PTS expressed significantly higher levels of proinflammatory cytokines in the cochlea (e.g., IL-1β, IL-6, and TNF-α). Overall, our findings suggest that cochlear microcirculation and lateral wall pathologies are differentially modulated by the severity of acoustic trauma and are associated with changes in vasoactive factors and inflammatory responses in the cochlea.

The dorsal (DCIC) and lateral cortices (LCIC) of the inferior colliculus are major targets of the auditory and non-auditory cortical areas, suggesting a role in complex multimodal information processing. However, relatively little is known about their functional organization. We utilized in vivo two-photon Ca2+ imaging in awake mice expressing GCaMP6s in GABAergic or non-GABAergic neurons in the IC to investigate their spatial organization. We found different classes of temporal responses, which we confirmed with simultaneous juxtacellular electrophysiology. Both GABAergic and non-GABAergic neurons showed spatial microheterogeneity in their temporal responses. In contrast, a robust, double rostromedial-caudolateral gradient of frequency tuning was conserved between the two groups, and even among the subclasses. This, together with the existence of a subset of neurons sensitive to spontaneous movements, provides functional evidence for redefining the border between DCIC and LCIC.

Sound localization in the horizontal plane (azimuth) relies mainly on binaural difference cues in sound level and arrival time. Blocking one ear will perturb these cues, and may strongly affect azimuth performance of the listener. However, single-sided deaf listeners, as well as acutely single-sided plugged normal-hearing subjects, often use a combination of (ambiguous) monaural head-shadow cues, impoverished binaural level-difference cues, and (veridical, but limited) pinna- and head-related spectral cues to estimate source azimuth. To what extent listeners can adjust the relative contributions of these different cues is unknown, as the mechanisms underlying adaptive processes to acute monauralization are still unclear. By providing visual feedback during a brief training session with a high-pass (HP) filtered sound at a fixed sound level, we investigated the ability of listeners to adapt to their erroneous sound-localization percepts. We show that acutely plugged listeners rapidly adjusted the relative contributions of perceived sound level, and the spectral and distorted binaural cues, to improve their localization performance in azimuth also for different sound levels and locations than those experienced during training. Interestingly, our results also show that this acute cue-reweighting led to poorer localization performance in elevation, which was in line with the acoustic–spatial information provided during training. We conclude that the human auditory system rapidly readjusts the weighting of all relevant localization cues, to adequately respond to the demands of the current acoustic environment, even if the adjustments may hamper veridical localization performance in the real world.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a regulatory and cytoprotective neuropeptide, its deficiency implies accelerated aging in mice. It is present in the auditory system having antiapoptotic effects. Expression of Ca 2+ -binding proteins and its PAC1 receptor differs in the inner ear of PACAP-deficient (KO) and wild-type (WT) mice. Our aim was to elucidate the functional role of PACAP in the auditory system. Auditory brainstem response (ABR) tests found higher hearing thresholds in KO mice at click and low frequency burst stimuli. Hearing impairment at higher frequencies showed as reduced ABR wave amplitudes and latencies in KO animals. Increase in neuronal activity, demonstrated by c-Fos immunolabeling, was lower in KO mice after noise exposure in the ventral and dorsal cochlear nuclei. Noise induced neuronal activation was similar in further relay nuclei of the auditory pathway of WT and KO mice. Based on the similar inflammatory and angiogenic protein profile data from cochlear duct lysates, neither inflammation nor disturbed angiogenesis, as potential pathological components in sensorineural hearing losses, seem to be involved in the pathomechanism of the presented functional and morphological changes in PACAP KO mice. The hearing impairment is probably concomitant with the markedly accelerated aging processes in these animals.

The gaseous free radical, nitric oxide ( NO ) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase ( nNOS ) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a subpopulation of each major neuronal type in the ventral cochlear nucleus ( VCN ). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L‐ NAME (L‐ NG ‐Nitroarginine methyl ester) and the NO donors SIN ‐1 (3‐Morpholinosydnonimine hydrochloride) and SNOG (S‐Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurones. Inhibition of endogenous NO production with L‐ NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L‐ NAME on NMDA ( N ‐Methyl‐D‐aspartic acid)‐evoked excitation, with 30% of units showing enhanced NMDA ‐evoked excitation during L‐ NAME application (reduced NO levels). Approximately 25% of neurones contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary‐like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss.

Spatial attention may be used to select target speech in one location while suppressing irrelevant speech in another. However, if perceptual resolution of spatial cues is weak, spatially focused attention may work poorly, leading to difficulty communicating in noisy settings. In electroencephalography (EEG), the distribution of alpha (8–14 Hz) power over parietal sensors reflects the spatial focus of attention [Banerjee, Snyder, Molholm, and Foxe (2011). J. Neurosci. 31, 9923–9932; Foxe and Snyder (2011). Front. Psychol. 2, 154.] If spatial attention is degraded, however, alpha may not be modulated across parietal sensors. A previously published behavioral and EEG study found that, compared to normal-hearing (NH) listeners, hearing-impaired (HI) listeners often had higher interaural time difference thresholds, worse performance when asked to report the content of an acoustic stream from a particular location, and weaker attentional modulation of neural responses evoked by sounds in a mixture [Dai, Best, and Shinn-Cunningham (2018). Proc. Natl. Acad. Sci. U. S. A. 115, E3286]. This study explored whether these same HI listeners also showed weaker alpha lateralization during the previously reported task. In NH listeners, hemispheric parietal alpha power was greater when the ipsilateral location was attended; this lateralization was stronger when competing melodies were separated by a larger spatial difference. In HI listeners, however, alpha was not lateralized across parietal sensors, consistent with a degraded ability to use spatial features to selectively attend.

Although information theoretic approaches have been used extensively in the analysis of the neural code, they have yet to be used to describe how information is accumulated in time while sensory systems are categorizing dynamic sensory stimuli such as speech sounds or visual objects. Here, we present a novel method to estimate the cumulative information for stimuli or categories. We further define a time-varying categorical information index that, by comparing the information obtained for stimuli versus categories of these same stimuli, quantifies invariant neural representations. We use these methods to investigate the dynamic properties of avian cortical auditory neurons recorded in zebra finches that were listening to a large set of call stimuli sampled from the complete vocal repertoire of this species. We found that the time-varying rates carry 5 times more information than the mean firing rates even in the first 100 ms. We also found that cumulative information has slow time constants (100-600 ms) relative to the typical integration time of single neurons, reflecting the fact that the behaviorally informative features of auditory objects are time-varying sound patterns. When we correlated firing rates and information values, we found that average information correlates with average firing rate but that higher-rates found at the onset response yielded similar information values as the lower-rates found in the sustained response: the onset and sustained response of avian cortical auditory neurons provide similar levels of independent information about call identity and call-type. Finally, our information measures allowed us to rigorously define categorical neurons; these categorical neurons show a high degree of invariance for vocalizations within a call-type. Peak invariance is found around 150 ms after stimulus onset. Surprisingly, call-type invariant neurons were found in both primary and secondary avian auditory areas.

The perception of a sound can be influenced by another sound in a space-dependent manner. An understanding of this perceptual phenomenon depends on knowledge about how the spatial relationship between two sounds affects neural responses to the sounds. We used the rat as a model system and equal-probability two-tone sequences as stimuli to evaluate how spatial separation between two asynchronously recurring sounds affected responses to the sounds in midbrain auditory neurons. We found that responses elicited by two tone bursts when they were colocalized at the ear contralateral to the neuron were different from the responses elicited by the same sounds when they were separated with one at the contralateral ear while the other at another location. For neurons with transient sound-driven firing and not responsive to stimulation presented at the ipsilateral ear, the response to a sound with a fixed location at the contralateral ear was enhanced when the second sound was separated. These neurons were likely important for detecting a sound in the presence of a spatially separated competing sound. Our results suggest that mechanisms underlying effects of spatial separation on neural responses to sounds may include adaptation and long-lasting binaural excitatory/inhibitory interaction.

Foxg1 is one of the forkhead box genes that are involved in morphogenesis, cell fate determination, and proliferation, and Foxg1 was previously reported to be required for morphogenesis of the mammalian inner ear. However, Foxg1 knock-out mice die at birth, and thus the role of Foxg1 in regulating hair cell (HC) regeneration after birth remains unclear. Here we used Sox2 CreER/+ Foxg1 loxp/loxp mice and Lgr5-EGFP CreER/+ Foxg1 loxp/loxp mice to conditionally knock down Foxg1 specifically in Sox2+ SCs and Lgr5+ progenitors, respectively, in neonatal mice. We found that Foxg1 conditional knockdown (cKD) in Sox2+ SCs and Lgr5+ progenitors at postnatal day (P)1 both led to large numbers of extra HCs, especially extra inner HCs (IHCs) at P7, and these extra IHCs with normal hair bundles and synapses could survive at least to P30. The EdU assay failed to detect any EdU+ SCs, while the SC number was significantly decreased in Foxg1 cKD mice, and lineage tracing data showed that much more tdTomato+ HCs originated from Sox2+ SCs in Foxg1 cKD mice compared to the control mice. Moreover, the sphere-forming assay showed that Foxg1 cKD in Lgr5+ progenitors did not significantly change their sphere-forming ability. All these results suggest that Foxg1 cKD promotes HC regeneration and leads to large numbers of extra HCs probably by inducing direct trans-differentiation of SCs and progenitors to HCs. Real-time qPCR showed that cell cycle and Notch signaling pathways were significantly down-regulated in Foxg1 cKD mice cochlear SCs. Together, this study provides new evidence for the role of Foxg1 in regulating HC regeneration from SCs and progenitors in the neonatal mouse cochlea.