Publications using TDT

Current disease-sensing devices primarily focus on distinguishing between healthy and diseased states, effective for diagnosis but limited in guiding optimal intervention timing for prevention. We developed a tripartite framework identifying pre-disease state in depression, a reversible phase preceding irreversible onset. Using complex systems theory, we analyzed early-warning signals emerging as biological systems approach critical transitions. Continuous monitoring of nine multimodal biomarkers—spanning electrophysiological, behavioral, and biological—enabled classification into normal, pre-disease, and disease states by quantitatively defining critical points. An artificial intelligence agent classified disease states with 95.2% accuracy using multimodal data, enabled by ultrasoft neural probes for stable, low-damage recordings. Therapeutic validation with a skin-attachable wireless vagus nerve stimulator integrating soft three-dimensional electrodes demonstrated superior efficacy during pre-disease states. Subjects treated during pre-disease showed faster recovery and greater therapeutic responses, while those treated after disease onset failed to achieve full recovery. This framework provides evidence-based rationale for early intervention.

Addicted individuals prefer drug rewards over natural rewards. Dysregulated dopamine (DA) signaling in the nucleus accumbens is thought to trigger this phenomenon, but there is little supporting evidence. Using a genetically encoded sensor, we monitored DA dynamics in mice in an operant associative task with natural (fat solution) or artificial (cocaine or optogenetic DA self-stimulation) rewards. During learning, DA transients emerged at predictive cues for both reward types, vanished at natural reward delivery, but persisted at artificial reward delivery. In choice sessions, animals’ preferences varied from exclusive natural reward to exclusive artificial reward. Individual preferences were predicted by cue-evoked DA response magnitudes. Revaluation of reward contingencies shifted reward-type preference and DA signaling accordingly, but not in mice showing compulsive drug seeking, indicating impaired value updating in these mice. These results support a model in which DA drives adaptive reward seeking, with persistent cue-associated DA signaling underpinning addiction vulnerability.

Activity of the mesolimbic dopamine system has long been implicated in encoding primary rewards and contributing to the addictive properties of drugs of abuse. Dopamine neurons in the ventral tegmental area (VTA DA ) of the midbrain typically show patterns of spontaneous burst activity that align with the onset of salient events or rewarding stimuli, resulting in phasic dopamine release in the nucleus accumbens (NAc). Fiber photometry is increasingly being used as an accessible technique to quantify neural activity with high temporal resolution at sensors offering signal specificity in stable recordings over extended periods of time. It has been well established by multiple techniques that opioids increase mesolimbic dopamine activity, likely through disinhibition of VTA DA neurons. Here we used fiber photometry to compare sub-second transient events from VTA DA neurons with GCaMP6f and dopamine release in the lateral shell of the NAc with dLight1.3b and GRABDA2h in response to morphine treatment. In weekly sessions, one dose of morphine was administered in escalating order (2.5, 5,7.5, and 10 mg/kg, intraperitoneal). Consistent with prior literature, both GCaMP6f in VTA DA neurons and dLight1.3b in NAc showed patterns of increased signal following morphine treatment. In contrast, morphine suppressed transient activity at GRABDA2h sensors. Further analyses of whole signal streams from each sensor showed a generalized increase, but reduction in variability of the GRABDA2h signal, consistent with the interpretation of sensor saturation. Such results emphasize the importance of the inclusion of appropriate controls to contextualize the interpretation of biosensor responses, particularly in response to pharmacological treatment. HIGHLIGHTS Morphine elicited increased signaling in VTA DA GCaMP6f and NAc dLight1.3b, consistent with prior literature Morphine suppressed NAc GRABDA2h signaling of transient events, suggesting saturation of GRABDA2h sensor Sensor validation with pharmacological challenges is critical for interpretation of data

Exposure to stress can cause long-lasting enhancement of fear and other defensive responses that extend beyond the cues or contexts associated with the original traumatic event. These nonassociative consequences of stress, referred to as fear sensitization, are thought to underlie some symptoms of trauma-related disorders. Fear sensitization has been predominantly studied using the stress-enhanced fear learning (SEFL) paradigm, which models the stress-induced amplification of fear learning. Less is known about the mechanisms through which unlearned fear responses are sensitized by stress. Here, we investigated the neural mechanisms for sensitization of unlearned fear responses using a paradigm we termed stress-enhanced fear responding (SEFR). In this model, mice exposed to a single session of footshock stress exhibit enhanced freezing to a novel tone stimulus. To investigate brain regions that might mediate SEFR, we first used c-Fos mapping to identify neural activity changes associated with stress-induced enhancement of unlearned fear. Our c-Fos screen identified the posterior paraventricular thalamus (pPVT) as a region that was persistently hyperactive after footshock stress and whose activity correlated with behavioral expression of SEFR. Using fiber photometry, we observed that SEFR, but not SEFL, was associated with increased activity in the pPVT. Next, we found that chemogenetic inhibition of the pPVT blocked both the induction of SEFR during stress and its later expression, while artificial stimulation of pPVT in stress-naive mice was sufficient to recapitulate SEFR. Interestingly, pPVT inhibition or stimulation did not affect acquisition or expression of SEFL. In conclusion, our results indicate that sensitization of fear learning (SEFL) and sensitization of unlearned fear (SEFR) have distinct neural mechanisms. Our results identify pPVT hyperactivity as a mechanism for stress-induced sensitization of unlearned fear and highlight pPVT as a potential target for treating arousal and reactivity symptoms of trauma- and stressor-related disorders.

Background Iron is a key nutrient for the development of the fetal auditory system. However, the potential impact of non-anemic prenatal iron deficiency (ID) on neonatal auditory function remains unclear. This study aimed to systematically explore the potential mechanisms by which maternal ID may affect auditory maturation of offspring. Methods We analyzed population data from 696 mother–infant pairs, established ID mouse models (C57BL/6 J) during pregnancy, and conducted cellular experiments. Results In the human cohort, maternal serum ferritin (SF) and hemoglobin (Hb) were significantly negatively associated with the latency (ms) of auditory brainstem response (ABR) waves I, III, and V, as well as intervals (ms) of waves I–III, III–V, and I–V and summating potential/action potential ratios (%). Neonatal SF partially mediated the association between maternal iron status and auditory function, with mediation effects ranging from 28.57 to 76.32%. In mouse models, prenatal ID was associated with decreased wave I amplitude and extended latency in offspring, along with reduced ribbon synapses in inner hair cells, mitochondrial damage, and decreased enzyme activity in supporting cells. A metabolomics analysis revealed significant downregulation of pyruvate levels in the ID group, and exogenous supplementation with sodium pyruvate partially restored ribbon synaptic function. Collectively, prenatal ID may reduce fetal iron reserves, impair energy metabolism of cochlear supporting cells, inhibit ribbon synaptic maturation, and potentially contribute to auditory dysfunction. Conclusion Our findings suggest that non-anemic maternal ID may be associated with delayed neonatal auditory maturation, highlighting the potential importance of iron intervention during pregnancy for improving neonatal auditory outcomes; however, causal relationships cannot be established from the observational human data, and the animal/cellular findings should be interpreted as supportive evidence requiring further validation.

The basolateral amygdala (BLA) is important for assigning emotional weight to environmental stimuli to guide learning, discrimination, and adaptive vigilance. The contribution of neuromodulator inputs such as dopamine to this process is unclear. Here, we recorded in vivo BLA dopamine signals across emotional learning paradigms. We show that BLA dopamine scales with a stimulus's emotional intensity, but not value or associative strength, and reward-predictive cues evoke BLA dopamine signals that diminish, rather than grow, with training. In a complex learning context, where rats discriminated between multiple valenced associations, the magnitude of cue-evoked BLA dopamine re-scaled to report the level of relative emotional saliency. Threat and safety cues prompted larger, sustained dopamine signals compared to reward and neutral cues. Together, our data reveal a pattern of dopamine encoding with a unique contribution to learning, for dynamic disambiguation of affective states.

Adult neurogenesis sustains olfactory function, facilitates the acquisition of new memories, and provides resilience against depression in mice. Neural stem cells (NSCs) elicit selective responses to different physiological and pathological stimuli, thereby modulating neuron production. Here, we show that fear conditioning stimulates neurogenesis in the adult mouse brain septum by activating NSCs in the dorsal septum (dsNSCs) situated near a plexus of serotonergic axons originating from raphe nucleus neurons—key regulators of mood, anxiety, and stress responses. Elevation of serotonin (5HT) levels with the antidepressant and anxiolytic drug fluoxetine similarly promoted dsNSC proliferation and neurogenesis. The adult-born GABAergic interneurons integrate into septal nuclei. These findings suggest a potential adaptive response in mice to stress-inducing aversive experiences and elevated 5HT in the adult septum.

Hearing impairment is attributed to factors such as age, genetic predisposition, and environmental influences, among which environmental factors are considered modifiable. Among various environmental factors, the role of poor lifestyle habits is particularly critical, yet the specific mechanisms by which they contribute to hearing damage remain unclear. This study reveals that dysregulated hormone levels due to disrupted light exposure may significantly increase susceptibility to sensorineural hearing loss. In mice, circadian rhythm disruption was found to reduce melatonin and elevate serotonin levels in the inner ear, thereby increasing vulnerability to cisplatin‐induced ototoxicity. In both in vivo and in vitro cisplatin‐treatment models, we showed that combined treatment with melatonin protected hearing, reduced inner ear cell death, and preserved synaptic connections, whereas serotonin co‐administration exacerbated the damage. Using small molecule–protein interaction prediction, we identified NOS3 as a potential target of both melatonin and serotonin, through which they appear to regulate the NO signaling pathway and influence hair cell ferroptosis. Finally, exogenous supplementation of NOS3 in cochlear tissues effectively mitigated cisplatin‐induced hair cell damage, even under conditions of circadian rhythm disruption. These findings indicate that the melatonin/serotonin balance modulates susceptibility to sensorineural hearing loss via the NOS3–NO signaling pathway.

Objective To evaluate the feasibility of using the acoustic change complex (ACC) as an objective cortical marker of continuous speech sound discrimination using connected Ling-six stimuli. Design Cortical auditory evoked potentials were recorded from 10 young adults with normal hearing using a continuous, pause-free sequence of the six Ling phonemes (/u-a-i-ʃ-s-m/). ACC responses were recorded in a passive listening paradigm with minimal electrodes. Reproducibility was assessed in a subset of participants retested 1 year later. Results Robust ACC responses were elicited for all six phonemic transitions in every participant, indicating reliable cortical discrimination within the continuous speech stream. Although response morphology varied across listeners, within-subject responses demonstrated strong reproducibility over a one-year interval (mean cross-correlation = 0.95). Conclusion Continuous Ling-six stimuli reliably evoke ACC responses reflecting cortical speech sound discrimination. This efficient and repeatable paradigm shows promise as an objective verification tool for auditory learning and sound discrimination, particularly in populations for whom behavioral assessment is unreliable.

In mammals, slow-wave sleep is characterized by synchronized neuronal activity that alternates between on and off periods. Slow-wave activity (SWA) and synchrony reflect sleep need, are correlated with synaptic strength in cortical circuits and promote synaptic downselection and memory consolidation. Here we assessed whether these core benefits of sleep can be obtained during waking. We locally induced alternating on/off periods during wakefulness using optogenetics in mice. This led to a local ipsilateral reduction in SWA and synchrony during subsequent sleep, and to reduced markers of synaptic strength. Moreover, bilateral induction of off periods over sensorimotor cortex during sleep deprivation restored memory consolidation. Thus, inducing on/off activity during wakefulness is sufficient to reduce local sleep need and fulfill core functions of sleep.

Noise-induced hearing loss (NIHL) is a major form of sensorineural hearing impairment driven by oxidative stress-mediated cochlear injury. Sanghuangporus sanghuang (SS), a medicinal fungus extensively studied in microbiology and biotechnology, is known to produce bioactive metabolites with antioxidant properties; however, its functional role in the auditory system has not been established. This study investigated the otoprotective potential of SS extract against oxidative and acoustic stress using complementary in vitro, ex vivo, and in vivo models. In H2O2-treated UB-OC1 auditory cells, SS (25-200 μg/mL) dose-dependently restored cell viability and significantly reduced intracellular reactive oxygen species accumulation. Western blot analysis demonstrated that SS suppressed the expression of apoptotic markers, including cleaved caspase-3 and cytochrome C. At the molecular level, SS upregulated Nrf2 and HO-1 expression at both mRNA and protein levels, without a significant change in Keap1 expression, indicating activation of endogenous antioxidant defense via the Nrf2/HO-1 signaling axis. Consequently, downstream antioxidant genes such as SOD1 and NQO1 were significantly upregulated. In ex vivo cochlear explant cultures, SS preserved hair cell integrity against H2O2-induced damage, as confirmed by phalloidin staining. In a murine NIHL model, oral administration of SS attenuated ABR threshold shifts, maintained Wave I amplitudes, and preserved the structural organization of outer hair cells across all cochlear turns. Collectively, these findings demonstrate that SS confers otoprotection by modulating the Nrf2/HO-1 antioxidant axis and mitigating oxidative stress-induced sensory cell injury, supporting the potential of SS as a fungal-derived functional bioactive resource for redox-associated cochlear protection.

Purpose: The UmboMic is a recently developed middle ear microphone that functions by detecting the sound-induced motion of the umbo [1]. To prepare for a live animal study of the in vivo performance of the UmboMic, we developed the surgical implantation and fixation system in cadaveric sheep ears. Methods: Temporal bones from seven female sheep were prepared for implantation of the UmboMic system. Fixation hardware for sheep was designed to secure the UmboMic sensor in position so that the sensing tip contacted the umbo in the middle-ear cavity and the non-sensing tail end was secured to the surrounding mastoid bone. First, pre-surgical micro-CT scans of the temporal bones and open-source software were used to simulate surgical drilling, and to virtually plan and fit the UmboMic fixation system. Then, in physical sheep temporal bones, sound-induced umbo motion was measured with a laser Doppler vibrometer as a preliminary step. After surgical implantation of the UmboMic system, the position of the UmboMic sensor was evaluated with microscopic visualization and post-surgical micro-CT. Implanted UmboMic sensor function in response to acoustic frequency sweeps to the external ear canal was measured in two specimens. Results: Measurements of sheep temporal bone anatomical dimensions with micro-CT showed that the facial recess featured a mean maximum height of 2.36 mm, length of 12.2 mm, and depth from outer opening surface of facial recess to the umbo of 11.1 mm. The amplitude of umbo motion prior to UmboMic implantation was consistent with that previously reported by [2], with a displacement of ~ 14 nm/Pa up to a frequency of 5.5 kHz, followed by a gradual drop-off. When bench tested and implanted in sheep temporal bones, the UmboMic sensitivity was as predicted from [1], ~ 1-2 fC/nm. Conclusion: When implanted in sheep cadaveric ears, UmboMic performance was similar to previous measurements in fresh human cadaveric temporal bones. The method of simulating surgical drilling using micro-CT and simple open-source software is economical and broadly applicable in understanding the 3D biological anatomy and implantable device design and customization. The UmboMic implantation in cadaveric sheep ears was possible, and this study represents a step towards planned live animal studies.

Adaptive behavior requires that behaviorally relevant signals gain access to neural circuits guiding action. The thalamus has long been proposed to regulate information flow to cortical and subcortical systems, yet whether it also tracks internally generated goal signals remains unclear. Here, we show that neurons in the paraventricular thalamus (PVT) projecting to the nucleus accumbens (NAc) encode the motivational value of safety during active avoidance. As mice learn to avoid threat, PVT→NAc neurons develop a signal that emerges selectively at successful avoidance, is experience-dependent, and diminishes following outcome devaluation. Selective silencing of the PVT→NAc pathway at safety onset reduces the motivational value assigned to safety without impairing action–outcome learning. Mechanistically, PVT input engages cholinergic interneurons (CIN) in the NAc to regulate dopamine release via synaptic potentiation mediated by GluA2-lacking AMPA receptors at PVT–CIN synapses. Disrupting this plasticity reduces the motivational impact of safety. These findings identify a thalamostriatal mechanism through which learned goals gain stable access to motivational circuitry.

CLN3 disease, the most common form of the Neuronal Ceroid Lipofuscinoses (NCLs), causes progressive cognitive decline and language impairment in humans. A pathological hallmark is the accumulation of storage material within neuronal lysosomes resulting from mutations in the CLN3 gene. We previously identified parallel deficits in auditory duration mismatch negativity (MMN), an electroencephalography (EEG)-based marker of auditory change detection, in individuals with CLN3 disease and in Cln3 -/- mice. MMN-dependent auditory change detection relies on sensory-memory comparison mechanisms. However, the anatomical and neurophysiological substrates underlying this response in CLN3 disease remain unclear. Here, we investigated central auditory dysfunction in Cln3 -/- mice by integrating immunohistochemical mapping of lysosomal storage pathology, using the canonical marker Subunit C of Mitochondrial ATP Synthase (SCMAS), with EEG analysis of auditory evoked potentials (AEPs). Neuropathological analyses revealed age-dependent and sex-divergent SCMAS accumulation across the auditory thalamocortical circuit, including the excitatory auditory thalamus, the inhibitory thalamic reticular nucleus, and the primary auditory cortex. In parallel, Cln3 -/- mice exhibited age- and sex-dependent alterations in AEPs relative to wild-type controls. Importantly, an integrated measure of auditory thalamocortical SCMAS accumulation accounted for a substantial portion of age- and sex-matched variation in AEP responses, with stronger associations for the early N1 component than the later MMN component. Together, these findings link age-dependent and sex-divergent auditory neurophysiological deficits to region-specific lysosomal storage pathology within the auditory thalamocortical circuit in the Cln3 -/- mouse model. This integrated functional-anatomical framework provides insight into circuit vulnerability and supports the development of translational neurophysiological biomarkers for CLN3 disease. Graphical Abstract Highlights SCMAS accumulates across the auditory thalamocortical circuit in Cln3 -/- mice Accumulation shows distinct age- and sex-dependent regional trajectories Cln3 -/- mice exhibit progressive alterations in auditory evoked potentials Thalamic pathology is strongly associated with the early auditory N1 response Histology–EEG integration links circuit pathology to auditory dysfunction

Mutations in Myo7a cause Usher syndrome type 1B and non-syndromic deafness, but the precise function of MYO7A in sensory hair cells remains unclear. Using long-read sequencing, we identify and characterize a novel isoform, MYO7A-N, expressed in auditory hair cells alongside the canonical MYO7A-C. Isoform-specific knock-in mouse models reveal that inner hair cells primarily express MYO7A-C, while outer hair cells express both isoforms in opposing tonotopic gradients. Both isoforms are localized to the upper tip-link insertion site, consistent with a role in the tip link for mechanotransduction. Loss of MYO7A-N leads to outer hair cell degeneration and progressive hearing loss. Cryo-EM structures reveal isoform-specific differences at actomyosin interfaces, correlating with distinct ATPase activities. These findings reveal an unexpected layer of molecular diversity within the mechanotransduction machinery. We propose that MYO7A isoform specialization enables fine-tuning of tip-link tension, thus hearing sensitivity, and contributes to the frequency-resolving power of the cochlea.

Acetylcholine (ACh) release from the basal forebrain has traditionally been viewed as a slow, spatially diffuse signal regulating cortical arousal across sleep and wakefulness 1–3 . Recent characterizations with higher resolution optical sensors have revealed rapid, local cholinergic modulation supporting dynamic changes in sensory processing, associative learning, and behavioral state 4–7 . However, sensory events that recruit cortical ACh often also change arousal and evoke movements, making it difficult to determine whether ACh transients reflect sensory features of environmental stimuli or the behavioral state changes that accompany sensory stimulation. To separate these contributions, we performed optic fiber recordings of a genetically encoded ACh fluorescent sensor in the auditory, visual, and prefrontal cortex of awake, head-fixed mice while monitoring pupil size and facial movements. Across cortical areas, ACh release tracked spontaneous fluctuations in arousal state, as indexed by pupil dilation and orofacial movements. Sensory stimuli also evoked rapid ACh transients, with sounds producing larger and more widespread responses than visual stimuli. Because sounds also elicited time-locked pupil dilations and facial movements, we used multivariate modeling to estimate the relative contributions of stimulus features, arousal, and behavior to cortical ACh dynamics. We identified a regional dissociation: sound-evoked ACh release in prefrontal cortex was largely explained by arousal- and movement-related variables, whereas auditory cortical ACh release retained a stronger relationship to stimulus features. These findings show that cortical ACh signaling reflects both shared arousal state and area-specific sensory processing and demonstrate that sound is especially effective at recruiting rapid, widespread cholinergic modulation across cortex.

Social recognition, the ability to distinguish between individuals, is essential for cognitively demanding social behaviors. The anterior olfactory nucleus (AON), a primary olfactory cortical region, is implicated in this process, but the underlying neurocircuitry remains poorly understood. Here, we generated a novel mouse line to enable genetic access to AON pyramidal neurons and mapped their whole-brain synaptic inputs and outputs. The medial prefrontal cortex (mPFC), a crucial hub for social cognition, is the primary neocortical target of AON neurons, which form monosynaptic excitatory connections with a substantial fraction of mPFC neurons. The AONmPFC pathway is activated during social investigation, and chemogenetic inhibition of this pathway impairs social recognition. Moreover, an analogous AON-prefrontal pathway is present in humans, as supported by resting-state functional magnetic resonance imaging (fMRI) functional connectivity analyses. Taken together, these findings reveal a conserved olfactory-prefrontal circuit spanning mice to humans, potentially linking olfactory dysfunction to neuropsychiatric disorders.

Auditory brainstem response (ABR) recordings are widely used in neuroscience as a cross-species translational tool to evaluate hearing sensitivity and auditory processing. In animal models, ABR recordings typically require the use of anesthesia to minimize myogenic artifacts, necessitating the development of anesthetic regimens that balance safety, adequate immobilization, and preservation of neural responses. Using a within-subject design in male and female Long-Evans rats, this study systematically evaluated the safety and auditory signal fidelity of five anesthetic regimens -ketamine/xylazine (K/X), ketamine/dexmedetomidine (K/D), dexmedetomidine alone (DEX), dexmedetomidine plus low dose (0.5%) isoflurane (D/I), and high dose (2%) isoflurane (ISO). Anesthetic safety and efficacy were assessed by induction time, recovery time, blood oxygen saturation (SpO₂), and survival rate. ABR signal quality was quantified by threshold, signal strength, wave amplitude, wave latency, and inter-trial phase coherence. K/X anesthesia produced ABRs with low thresholds and well-defined waveforms, but also exhibited the least favorable safety profile, with prolonged recovery times and reduced survival rates. Conversely, isoflurane regimens (ISO and D/I) had a favorable safety profile but significantly elevated thresholds and degraded ABR signal quality, even at low doses. K/D and DEX achieved survival and recovery outcomes equal to or better than conventional anesthetics (K/X and ISO) while also preserving ABR waveform morphology and trial-to-trial reliability. Together, these results highlight dexmedetomidine, particularly when combined with ketamine, as a safe and effective anesthetic for ABR recordings that preserves key metrics of auditory sensitivity and signal integrity.

Preparing and initiating movements at the right time is critical for goal-directed behavior. Before movement execution, motor cortical areas exhibit preparatory activity, which decreases from premotor to primary motor areas. During the shift from preparation to execution activity in neural state space transitions from movement-null to movement-potent dimensions. However, the circuit-level mechanisms underlying this shift remain unresolved. Here, we demonstrate that projections from the rat premotor cortex (rostral forelimb area [RFA]) to the primary motor cortex (caudal forelimb area [CFA]) encode primarily pre-movement activity. Optogenetic inhibition of these projections has behavioral effects comparable to inhibiting either RFA or CFA alone. During preparation, RFA projections enhance and suppress CFA neurons similarly, affecting activity along CFA's preparatory dimension. During movement, RFA's influence shifts predominantly to excitatory, aligning with CFA's movement-potent dimension. These results establish a mechanistic link between neural state space concepts and underlying circuit mechanisms, providing an intuitive model for movement control.

Kanamycin (KM), a widely used aminoglycoside antibiotic, is limited by its ototoxic side effects, primarily targeting cochlear outer hair cells (OHCs) and leading to irreversible sensorineural hearing loss. Our previous work showed that silencing AMP‐activated protein kinase alpha 1 (AMPKα1) prevents noise‐induced hearing loss through a redox‐sensitive regulatory mechanism. Given that both noise‐induced and aminoglycoside‐induced hearing loss share oxidative stress as a key pathological feature, we tested whether AMPKα1 silencing could mitigate KM‐induced damage using an acute ototoxic model combining KM with furosemide (KM + FU). KM + FU exposure markedly increased AMPKα phosphorylation at Thr172. Targeted siRNA‐mediated silencing via posterior semicircular canal delivery in CBA/J mice significantly reduced AMPKα1 expression in OHCs and effectively prevented KM + FU‐induced OHC loss and hearing impairment. This preventive effect was independent of stria vascularis permeability or cochlear KM uptake. These findings identify AMPKα as a contributor to KM + FU‐induced acute ototoxicity and suggest that its inhibition may be a promising strategy for hearing preservation.