Behavioral Neuroscience publications

Long-term sustained pain following acute physical injury is a prominent feature of chronic pain conditions1. Populations of neurons that rapidly respond to noxious stimuli or tissue damage have been identified in the spinal cord and several nuclei in the brain2-4. Understanding the central mechanisms that signal ongoing sustained pain, including after tissue healing, remains a challenge5. Here we use spatial transcriptomics, neural manipulations, activity recordings and computational modelling to demonstrate that activity in an ensemble of anatomically and molecularly diverse parabrachial neurons that express the neuropeptide Y (NPY) receptor Y1 (Y1R neurons) is increased following injury and predicts functional coping behaviour. Hunger, thirst or predator cues suppressed sustained pain, regardless of the injury type, by inhibiting parabrachial Y1R neurons via the release of NPY. Together, our results demonstrate an endogenous analgesic hub at pain-responsive parabrachial Y1R neurons.

Discrimination of sensory stimuli is fundamentally constrained by the information encoded in neuronal responses. In the barn owl, interaural time difference (ITD) serves as a primary cue for azimuthal sound localization and is represented topographically in the midbrain auditory space map in the external nucleus of the inferior colliculus (ICx). While prior studies have demonstrated a correspondence between spatial tuning and behavioral acuity, it remains unclear how changes in sensory reliability influence this relationship. Here, we examined how behavioral and neuronal ITD discrimination thresholds vary with binaural correlation (BC), which manipulates ITD cue reliability. Using the pupil dilation response as a behavioral metric in head-fixed owls of either sex, we found that ITD just-noticeable differences increased exponentially as BC decreased. In contrast, the widths of ICx ITD tuning curves increased more modestly, indicating that tuning resolution alone does not account for behavioral discrimination performance. By computing the Fisher information from ICx neuronal responses, we showed that the average neuronal discriminability predicts behavioral thresholds across BC values. A habituation-based model incorporating BC-dependent changes in tuning width, firing rate, and response variability successfully accounted for both direction and ITD discrimination. These findings support a model in which perceptual acuity is governed by the combined influence of neuronal tuning and variability and provide a unified framework for understanding how midbrain auditory representations underlie adaptive spatial hearing.

Spiny projection neurons (SPNs) in the dorsal striatum play crucial roles in locomotion control and value-based decision-making. SPNs, which include both direct-pathway striatonigral and indirect-pathway striatopallidal neurons, can be further classified into subtypes based on distinct transcriptomic profiles and cell body distribution patterns. However, how these SPN subtypes regulate spontaneous locomotion in the context of environmental valence remains unclear. Using Sepw1-Cre transgenic mice, which label a specific SPN subtype characterized by a patchy distribution of cell bodies in the dorsal striatum, we found that these patchy striatonigral neurons constrain motor vigor in response to valence differentials. In a modified light/dark box test, mice exhibited differential walking speeds between the light and dark zones. Genetic ablation of these patchy SPNs disrupted restful slowing in the dark zone and increased zone discrimination by speed. In vivo recordings linked the activity of these neurons to zone occupancy, speed, and deceleration, with a specific role in mediating deceleration. Furthermore, chemogenetic activation of patchy SPNs—and optical activation of striatonigral neurons in particular—reduced locomotion and attenuated speed-based zone discrimination. These findings reveal that a subtype of patchy striatonigral neurons regulates implicit walking speed selection based on innate valence differentials.

Basolateral amygdala (BLA) GABAergic interneurons (INs) are critical for associative learning; however, the comparative synaptic organization and learning-related activity of BLA IN types have not been systematically evaluated. Here, we show somatostatin (SOM) INs provide inhibition onto, and are excited by, local pyramidal cells (PCs), whereas vasoactive intestinal peptide (VIP) INs are driven by extrinsic afferents. Parvalbumin (PV) INs inhibit PCs and are activated by local and extrinsic inputs. Thus, SOM and VIP INs exhibit complementary roles in feedback and feedforward inhibition, respectively, while PV INs contribute to both microcircuit motifs. Functionally, each IN subtype exhibits unique activity patterns across fear and extinction learning, with SOM and VIP INs showing most divergent characteristics, and PV INs display an intermediate phenotype. Finally, SOM and PV INs dynamically track behavioral-state transitions across learning. These data provide insight into the synaptic microcircuit organization and activity of distinct BLA IN classes across aversive associative learning.

Introduction The posterior tail of the striatum receives dense inputs from sensory regions of cortex and thalamus, as well as midbrain dopaminergic innervation, providing a neural substrate for associative sensory learning. Previously, we have demonstrated that developmental hearing loss is associated with aberrant physiological states in striatal medium spiny neurons (MSNs). Methods Here we directly investigated auditory associative learning impairments in the striatum of adult Mongolian gerbils that underwent transient developmental hearing loss or sham hearing loss during the critical period of auditory development. We used electrophysiology to reveal significant changes to neuronal population responses in vivo and intrinsic and synaptic properties to medium spiny neurons in vitro as animals learned an appetitive “Go/No-Go” auditory discrimination task. For in vivo experiments a 64-channel electrode was implanted in the auditory region of the posterior tail of the striatum and neuronal recordings were carried out as animals learned the task. For in vitro experiments, corticostriatal slice preparations were made from animals on each day of training. Results In naïve animals from both groups there was limited to no phase locking to either auditory stimulus in vivo , and long term depression resulted from theta burst stimulation in vitro . Furthermore, intrinsic and synaptic properties in normal hearing animals were unaffected; however, the hearing loss group continued to show lowered synaptic inhibition, synaptic hyperexcitation, and suppressed intrinsic excitability in the hearing loss group. Starting around day 3–4 in both groups, the emergence of striatal medium spiny neuron phase locking to the auditory conditioning stimuli was observed in vivo . This occurred contemporaneous to an increased probability of theta burst induced LTP during MSN whole cell recording in vitro , and acquisition of the task as the correct rejection response significantly increased in the behaving animals. During the acquisition phase MSNs in the normal hearing group showed a significant decrease in synaptic inhibition and increase in synaptic excitation with no change to intrinsic excitability, while the MSNs in the hearing loss group showed a significant increase in synaptic inhibition, reduction of synaptic hyper excitability, and compensatory changes to intrinsic excitability that supported normal action potential generation. In both groups, synaptic properties were resolved to similar level of E/I balance that could be part of a conserved learning state. Discussion These changes to the intrinsic and synaptic properties likely support LTP induction in vivo and the strengthening of synapses between auditory inputs and MSNs that facilitate neuronal phase locking. These findings have significant implications for our understanding of striatal resilience to sensory impairments in early life, in addition to establishing a granular understanding of the striatal circuit changes that support reward driven stimulus–response learning.

Speech comprehension, especially in the presence of background sounds, allegedly declines as a consequence of noise-induced hearing loss. However, the connection between noise overexposure and deteriorated speech-in-noise perception despite normal audiometric thresholds (hidden hearing loss) is not yet clear. This study investigates speech-in-noise discrimination in young-adult Mongolian gerbils before and after an acoustic trauma to examine the link between noise exposure and speech-in-noise perception. Nine young-adult gerbils were trained to discriminate a deviant consonant-vowel-consonant combination (CVC) or vowel-consonant-vowel combination (VCV) in a sequence of CVC or VCV standards, respectively. The logatomes were spoken by different speakers and masked by a steady-state speech-shaped noise. After the gerbils obtained the behavioral baseline data, they underwent an acoustic trauma and participated in the behavioral experiments again. Applying multidimensional scaling, response latencies were used to generate perceptual maps reflecting the gerbils’ internal representations of the sounds pre- and post-trauma. To evaluate how the discrimination of vowels and consonants was altered after noise exposure, changes in response latencies between phoneme pairs were investigated in relation to their articulatory features. Numbers of intact inner hair cell synapses were counted, and auditory brainstem responses were measured to assess peripheral auditory function. Perceptual maps of vowels and consonants were very similar before and after noise exposure. Interestingly, the gerbils’ overall vowel discrimination ability was improved after the acoustic trauma, even though the gerbils suffered from noise-induced hearing loss with a temporary threshold shift for frequencies above 4 kHz. In contrast, there were only minor changes in the gerbils’ consonant discrimination ability. Moreover, noise exposure showed a differential influence on response latencies for vowel and consonant discriminations depending on the articulatory features. Altogether, the results show that an acoustic trauma followed by a temporary threshold shift is not necessarily linked to speech-in-noise perception difficulties associated with hidden hearing loss.

In the hippocampal formation, cholinergic modulation from the medial septum/diagonal band of Broca is known to correlate with the speed of an animal's movements at subsecond timescales and also supports spatial memory formation. Yet, the extent to which subsecond cholinergic dynamics, if at all, align with transient behavioral and cognitive states supporting the encoding of novel spatial information remains unknown. In this study, we used fiber photometry to record the temporal dynamics in the population activity of septo-hippocampal cholinergic neurons at subsecond resolution during a hippocampus-dependent object location memory task using ChAT-Cre mice of both sexes. Using a linear mixed-effects model, we quantified the extent to which cholinergic dynamics were explained by changes in movement speed; behavioral states such as locomotion, grooming, and rearing; and hippocampus-dependent cognitive states such as recognizing a novel location of a familiar object. The data show that cholinergic dynamics contain a multiplexed code of fast and slow signals (1) coding for the logarithm of movement speed at subsecond timescales, (2) providing a phasic spatial novelty signal during the brief periods of exploring a novel object location, and (3) coding for recency of environmental change at a seconds-long timescale. Furthermore, behavioral event-related phasic cholinergic activity demonstrates that fast cholinergic transients correlate with a switch in cognitive and behavioral states. These findings enhance understanding of the mechanisms by which cholinergic modulation contributes to the coding of movement speed and encoding of novel spatial information.

Early sensory experience can exert lasting perceptual consequences. For example, a brief period of auditory deprivation early in life can lead to persistent spatial hearing deficits. Some forms of hearing loss (i.e., conductive; CHL) can distort acoustical cues needed for spatial hearing, which depend on inputs from both ears. We hypothesize that asymmetric acoustic input during development disrupts auditory circuits that integrate binaural information. Here, we identify prolonged maturation of the binaural auditory brainstem in the guinea pig by tracking auditory evoked potentials across development. Using this age range, we induce a reversible unilateral CHL and ask whether behavioral and neural maturation are disrupted. We find that developmental CHL is associated with alterations in a brainstem readout of binaural function, an effect that was not observed in a separate cohort with adult-onset CHL. Startle-based behavioral measures suggest that Early CHL animals exhibit reduced spatial resolution for high-frequency sound sources. Finally, single-unit recordings of auditory midbrain neurons reveal significantly poorer neural acuity to a sound location cue that largely depends on high-frequency sounds. Thus, these findings show that unilateral deprivation can disrupt developing auditory circuits that integrate binaural information and may give rise to lingering spatial hearing deficits.

Behavioural adaptations to environmental threats are crucial for survival1,2 and necessitate rapid deployment of energy reserves3-5. The amygdala coordinates behavioural adaptations to threats6, but little is known about its involvement in underpinning metabolic adaptations. Here we show that acute stress activates medial amygdala (MeA) neurons that innervate the ventromedial hypothalamus (MeAVMH neurons), which precipitates hyperglycaemia and hypophagia. The glycaemic actions of MeAVMH neurons occur independently of adrenal or pancreatic glucoregulatory hormones. Using whole-body virus tracing, we identify a polysynaptic connection from MeA to the liver that promotes the rapid synthesis of glucose by hepatic gluconeogenesis. Repeated stress exposure disrupts MeA control of blood glucose, resulting in diabetes-like dysregulation of glucose homeostasis. Our findings reveal an amygdala-liver axis that regulates rapid glycaemic adaptations to stress and links recurrent stress to metabolic dysfunction.

The treatment of inner and middle ear diseases remains a significant challenge, often requiring surgical intervention as the only option. In this study, we investigated porphysomes, self-assembled porphyrin-based nanoparticles, as a minimally invasive drug delivery platform for inner ear applications and as photothermal agents for cholesteatoma ablation. Three porphysome formulations were evaluated: parent porphysomes (PS), porphyrin-stabilized nanoemulsions (nPS), and EDTA-lipid incorporated porphysomes (ePS). Rats received intratympanic injections of each formulation, and fluorescence imaging performed 1 h postinjection demonstrated concentration-dependent inner ear penetration for all formulations. Notably, no signs of ototoxicity were observed based on histological and functional assessments of hearing and balance. Among ePS-treated rats, the 250 and 1,000 μM groups showed significantly higher inner ear fluorescence compared with the 50 μM group. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) tests were performed at baseline and 2 and 6 weeks postinjection. No significant threshold shifts were detected in PS and ePS groups compared with controls. In contrast, nPS-treated rats exhibited a significant ABR threshold elevation at 6 weeks ( p < 0.02). At 6 weeks, minor yet statistically significant DPOAE threshold differences were observed at 16 kHz (PS group) and 32 kHz (all porphysome groups); however, all shifts remained below 0 dB, indicating no functional hearing loss. Vestibular assessments, including swim and beam tests, revealed no significant impairments. Upon laser activation, all porphysome formulations induced substantial temperature elevation (Δ T = 31 ± 3.76°C, p < 0.05) and histological burn effects in cholesteatoma tissues. These findings support the potential of porphysomes as a safe, minimally invasive drug delivery system and photothermal agent for the treatment of inner and middle ear disorders.

Internal motive states, such as sexual arousal, drive behaviour in response to social cues. However, little is known about how internal states and external cues are integrated to release appropriate behaviours at the correct moment during a social interaction, such as the transition from the appetitive to the consummatory phases of mating 1,2 . Here we identify a neural circuit in male mice that gates the onset of consummatory reproductive behaviours on contact with a mating partner. Stimulating MPOA Esr1 ∩ Vgat hypothalamic neurons promotes mounting of conspecifics and three-dimensional dummy objects 3 . We find that such mounting depends on mechanosensory but not visual cues. Through a large-scale electrophysiological screen, we identify neurons in the subparafascicular thalamic nucleus that nonlinearly integrate medial preoptic area of the hypothalamus (MPOA) and mechanosensory input to encode contact with a potential mate. Circuit tracing and perturbations demonstrated that this conjunctive coding occurs by means of convergent disinhibition from MPOA and excitation from the spinal trigeminal nucleus. Functional manipulations and calcium recordings showed these social-contact neurons, marked by parathyroid hormone 2, were essential for and able to promote mounting. These data indicate that subparafascicular thalamic nucleus-parathyroid hormone 2 neurons integrate internal drive with social touch to trigger mounting at opportune moments during mating. More generally, our findings uncover a brain mechanism whereby an internal state can attribute a social quality to a generic touch to initiate purposeful reproductive actions.

Social isolation enhances sociability, suggesting that social behavior is maintained through a homeostatic mechanism. Further, mammalian social needs shift dramatically from infancy through adolescence into adulthood, raising the question of whether the neural mechanisms governing this homeostatic regulation evolve across developmental stages. Here, we show that agouti-related peptide (Agrp) neurons in the arcuate nucleus of the hypothalamus, which are known to drive hunger in adults, are activated by social isolation from weaning through adolescence but not in adulthood. Importantly, the activity of these neurons is critical for social behavior during adolescence: inhibiting Agrp neurons reduced isolation-induced sociability in juveniles, but not in adults, and Agrp neuron activation promoted sociability only in young mice. After isolation, reunion with siblings or other conspecifics, but not unfamiliar adult males or amicable rat pups, rapidly decreased neuronal activity in juveniles, an effect requiring intact olfaction. These findings identify Agrp neurons as a key component of the circuitry governing age-specific social homeostasis.

The primary auditory cortex (ACtx) extracts behaviorally relevant information from complex sounds and supports the categorization of continuous acoustic features into discrete perceptual or behavioral classes. Although ACtx is known to be necessary for certain sound-guided behaviors, how its contributions evolve during learning remains unclear. This evolving role may be shaped by the diversity of its excitatory output pathways, which include multiple classes of projection neurons that transmit sensory information to downstream targets. To address this question, we trained head-fixed mice to categorize the temporal rate of sinusoidally amplitude-modulated (sAM) noise and used cell-type-specific optogenetic inhibition to assess ACtx function across learning. Silencing the ACtx impaired performance throughout learning and altered decision strategies by inducing a fluctuating choice bias and increasing reliance on prior choices, without affecting motor output. Inhibiting intratelencephalic (IT) or extratelencephalic (ET) projection neurons on just 20% of trials significantly impaired learning but had only a modest effect on expert performance. These findings demonstrate that the ACtx supports both the acquisition and expression of categorical behavior, with IT and ET projection pathways playing important roles in learning-related plasticity.

To survive, animals must rapidly learn to avoid predictable threats. Such learning depends on detecting reliable cue-outcome relationships that efficiently drive behavioral adaptations. The medial prefrontal cortex (mPFC) integrates learned information about the environment to guide adaptive behaviors1,2,3,4,5,6,7 and is critical for threat avoidance.8,9,10,11,12,13,14 However, most studies focused on well-learned threat avoidance strategies, and the specific inputs that signal avoidability and drive rapid avoidance learning remain poorly understood. Dopamine (DA) inputs from the ventral tegmental area (VTA) potently modulate prefrontal function and are preferentially engaged by aversive stimuli.15,16,17,18,19,20,21 Pharmacological blockade, DA depletion, and microdialysis experiments implicated DA in threat avoidance22,23,24,25 but lacked the spatiotemporal resolution required to define the timing of mPFC DA signals during avoidance learning. We used high-resolution tools to dissect the role of the VTA-mPFC DA circuit in rapid avoidance learning. Optogenetic suppression of VTA DA terminals in mPFC selectively slowed learning of a cued avoidance response without affecting cue-shock association learning, reactive escape behaviors, or expression of previously learned avoidance. Using a fluorescent DA sensor, we observed rapid, event-locked DA activity that emerged transiently during learning initiation. Increased DA encoded aversive outcomes and their predictive cues, while decreased DA encoded their omission and predicted how quickly mice learned to avoid. In yoked mice lacking control over shock omission, these dynamics were largely absent. Together, these findings demonstrate that the VTA-mPFC DA circuit is necessary for rapid acquisition of proactive avoidance behaviors and reveal transient event-related DA signals underlying this form of learning.

Nicotine is one of the most widely used addictive substances, yet its primary reinforcing effects are relatively weak. Nicotine's ability to potentiate responding for conditioned reinforcers is thought to drive persistent drug use. Here, we show that nicotine failed to alter responding maintained by a primary sucrose reinforcer (under a variable-ratio [VR] 11 schedule) across a broad dose range (0.01-1.0 mg/kg). Yet, nicotine enhanced operant behavior under a second-order reinforcement schedule in which responses produced sucrose-associated cues and were only intermittently reinforced by sucrose itself. Together, we demonstrate that nicotine selectively augments behavior maintained by conditioned reinforcers (sucrose-associated cues) in a rate-dependent manner, increasing responding only in mice with low rates of reinforcement behavior at baseline. Using fiber photometry, we demonstrate that nicotine selectively amplified cue-evoked dopamine release in the dorsolateral striatum-but only in low baseline responders-while having no effect on dopamine signaling in the nucleus accumbens. These effects were blocked by the nicotinic receptor antagonist mecamylamine. Further, nicotine's influence on behavior was abolished when the contingency between action and conditioned stimuli was disrupted, indicating that nicotine strengthens cue control of behavior rather than increasing motivation generally. Collectively, these findings reveal that nicotine's behavioral actions emerge through an interaction between pharmacological mechanisms, behavioral contingencies, and individual differences in baseline behavioral control. SIGNIFICANCE STATEMENT: Nicotine strengthens the impact of environmental cues on behavior by amplifying dopamine signals in specific projection targets, but only in individuals with specific behavioral traits. This reveals how nicotine hijacks learning processes to promote persistent, cue-driven actions.

Alcohol seeking during abstinence is mediated in part by strong associations between the pharmacological effects of alcohol and the environment within which alcohol is administered. The amygdala, particularly the basolateral amygdala (BLA), is a key neural substrate of environmental cue and reward associations since it is involved in associative learning and memory recall. However, we still lack a clear understanding of how alcohol affects the activity of BLA neurons, which may encode information that drives environmental cue-dependent, alcohol-related behaviors. We previously demonstrated that a subset of BLA neurons which express the calcium/calmodulin-dependent protein kinase II (CaMKII) and thymus cell antigen 1 (Thy1) markers project preferentially to the nucleus accumbens (NAcc), rather than the central amygdala; and these neurons mediate fear inhibition rather than fear acquisition or expression, suggesting a specific role in positive valence processing. We now demonstrate that Pavlovian conditioning with alcohol administration increases the activity of these Thy1-expressing (Thy1+) excitatory neurons in mouse BLA, which is necessary for the conditioned appetitive response. In vivo calcium imaging indicates that the temporal activity profile of these neurons is also correlated with alcohol-induced motivated behavior in response to environmental cues. Optogenetic inhibition of BLA Thy1+ neuronal activity at cell body disrupts both the formation and expression of alcohol-induced conditioned place preference. Furthermore, selective axonal inhibition of BLA-Thy1+ efferents reveals that the activity of their NAcc and prefrontal cortex (PFC) projections are differentially necessary for alcohol cue association vs. recall, respectively. Together, these findings provide insights into a molecularly distinct subset of BLA neurons that regulates environmental cue-reward associations and drives alcohol-induced motivated behaviors in a projection-specific manner.

The ability to recognize speakers by their voice despite acoustical variation plays a substantial role in primate social interactions. Although neurons in the macaque anterior temporal lobe (ATL) show invariance to face viewpoint, whether they also encode abstract representations of caller identity is not known. Here, we demonstrate that neurons in the voice-selective ATL of two macaques support invariant voice identity representations through dynamic population-level coding. These representations minimize neural distances across different vocalizations from the same individual while preserving distinct trajectories for different callers. This structure emerged from the coordinated activity of the broader neuronal ensemble, although a small subset of highly identity-selective neurons carried high identity information. Our findings provide a neural basis for voice identity recognition in primates and highlight the ATL as a key hub for integrating perceptual voice features into higher-level identity representations.

Chemical feedback is ubiquitous in physiology but is challenging to study without perturbing basal functions. One example is addictive drugs, which elicit a positive-feedback cycle of drug-seeking and ingestion by acting on the brain to increase dopamine signalling 1–3 . However, interfering with this process by altering basal dopamine also adversely affects learning, movement, attention and wakefulness 4 . Here, inspired by physiological control systems, we developed a highly selective synthetic physiology approach to interfere with the positive-feedback cycle of addiction by installing a cocaine-dependent opposing signalling process into this body–brain signalling loop. We used protein engineering to create cocaine-gated ion channels that are selective for cocaine over other drugs and endogenous molecules. Expression of an excitatory cocaine-gated channel in the rat lateral habenula, a brain region that is normally inhibited by cocaine, suppressed cocaine self-administration without affecting food motivation. This artificial cocaine-activated chemogenetic process reduced the cocaine-induced extracellular dopamine rise in the nucleus accumbens. Our results show that cocaine chemogenetics is a selective approach for countering drug reinforcement by clamping dopamine release in the presence of cocaine. In the future, chemogenetic receptors could be developed for additional addictive drugs or hormones and metabolites, which would facilitate efforts to probe their neural circuit mechanisms using a synthetic physiology approach. As these chemogenetic ion channels are specific for cocaine over natural rewards, they may also offer a route towards gene therapies for cocaine addiction.

Behavioral outcomes are rarely certain, requiring subjects to discriminate between available choices by using feedback to guide future decisions. Probabilistic reversal learning (PRL) tasks test subjects’ ability to learn and flexibly adapt to changes in reward contingencies. Cortico-striatal circuitry has been broadly implicated in flexible decision-making—though what role these circuits play remains complicated. In this study we leveraged the fast temporal dynamics of local field potentials to precisely identify the role that cortico-striatal networks play during PRL reward feedback. We measured widespread (32-CH) local field potential activity of male Long–Evans rats during a PRL task wherein a target response delivered reward on 80% of trials while a non-target response delivered reward on 20% of trials. When subjects learned those reward probabilities, contingencies were reversed. We found that reward-evoked oscillations at beta (15–30 Hz) and high gamma (>70 Hz) frequencies marked positive reward valence and reflected probability of reward. Activity and connectivity at beta frequencies between orbitofrontal cortex, anterior insula, medial prefrontal cortex, and ventral striatum during expected rewards were correlated with behavioral performance and specific aspects of value/exploitative behavior as defined by a reinforcement learning computational model. Finally, we found that modulating beta activity in orbitofrontal cortex with optogenetic (20 Hz) stimulation promoted maladaptive behavior when stimulation was provided during non-target responses, consistent with our data and computational model predictions. Reward-evoked beta oscillations may reflect a crucial component underlying reward learning, and erroneous elevations in this physiological signal may contribute to maladaptive task performance and behavioral disruptions.

Adeno-associated virus-based gene therapy offers a promising treatment paradigm for inner ear diseases; however, the genetic heterogeneity of hereditary deafness requires gene-specific strategies and optimization of current approaches to identify the range of treatable conditions and improve therapeutic outcomes. To consider the therapeutic potential for a hearing loss gene not previously explored, we investigated the gene encoding the chloride intracellular channel protein CLIC5, mutations in which lead to DFNB103 in humans and deafness and circling behavior in a Clic5 -deficient mouse model. In this study, we utilized two constructs to deliver the wild-type Clic5 coding sequence into Clic5 -deficient mice: single-stranded and self-complementary adeno-associated virus, the latter known for rapid onset of transgene expression. We report a robust restoration of CLIC5 expression using either construct, including prevention of morphological degeneration and preserving auditory and vestibular function. Interestingly, the self-complementary construct achieved comparable functional recovery to single-stranded construct but at a lower titer. These findings highlight the potential of self-complementary adeno-associated virus to reduce dose requirements, minimize toxicity and broaden clinical utility for inner ear therapies.