Auditory Neurophysiology publications

Optogenetic stimulation is widely used to establish causal links between neural activity, behavior, and sensory perception, yet animals may also perceive the neural stimulation itself, a phenomenon termed “optoception.” We hypothesized that cortical optogenetic stimulation would generate an artificial percept that could be distinguished from externally evoked sensations and characterized psychometrically in an operant task. We tested this hypothesis using mesoscale cortical photostimulation delivered by a 3 mm, 459 nm LED implanted over auditory cortex. Four mice expressing Channelrhodopsin-2 (ChR2) in excitatory neurons learned a head-fixed go/no-go task in which 20 Hz cortical photostimulation served as the target. ChR2-positive mice detected cortical photostimulation relative to LED-off catch trials, distinguished it from both an external LED matched in wavelength and pulse rate and from an acoustic noise matched in amplitude-modulation rate, and discriminated between 20- and 10-Hz cortical photostimulation. Reducing implanted-LED optical power initially lowered rate-discrimination sensitivity, which then increased across training sessions, demonstrating perceptual learning. In two mice tested with 11 randomly interleaved rates spanning 10-20 Hz, lick probability followed a saturating rate-dependent function. Two ChR2-negative littermates failed to acquire photostimulation-locked behavioral responding. Together, these findings show that mesoscale cortical optogenetic stimulation generates a graded, learnable artificial percept whose behavioral readout depends on photostimulation rate and optical power. This single-LED preparation provides a cost-efficient and wavelength-extensible platform for psychometric analysis of optoception. SIGNIFICANCE STATEMENT Optogenetic stimulation can alter sensory processing, but the neural perturbation itself may also become a perceptual cue. We show that mice detect mesoscale cortical photostimulation delivered by a 3 mm implanted LED, distinguish it from matched external light and sound, and discriminate differences in photostimulation rate. When implanted-LED optical power was reduced, discrimination sensitivity initially declined and then increased across training sessions, revealing perceptual learning of optoception. Thus, mesoscale cortical photostimulation generates a graded internal signal whose behavioral readout depends on stimulation rate, optical power, and experience. A single implanted LED provides a cost- efficient and wavelength-extensible assay for measuring optogenetic perception.

Cochlear damage triggers compensatory primary auditory cortex (A1) plasticity that amplifies responses to residual sensory inputs, thereby contributing to the restoration of both cortical responsiveness to sound and perceptual sound detection threshold. However, this adaptation can become maladaptive, producing neuronal hyperactivity that contributes to tinnitus and hyperacusis. The neuromodulatory mechanisms governing these adaptive and maladaptive changes remain unknown. Here, we demonstrate that noise-induced cochlear injury triggers bidirectional synaptic zinc signaling plasticity that potentiates activity in excitatory principal neurons and parvalbumin-expressing interneurons, while suppressing activity in somatostatin-expressing interneurons. These cell-type-specific effects of synaptic zinc plasticity contribute to restoring A1 responsiveness to sound and perceptual detection thresholds, while being necessary for neural hyperactivity. Together, our findings establish synaptic zinc as a pivotal neuromodulator that shapes both adaptive and maladaptive cortical plasticity and identify a promising therapeutic target for improving perceptual recovery after cochlear damage and mitigating tinnitus and hyperacusis.

Through learning and memory, the cortex dynamically forms sensory associations to guide our behavior in an ever-changing environment. How memory-related brain regions, such as the medial-temporal lobe, influence sensory cortex to support this process remains unclear. Here, we used two-photon calcium imaging of axonal projections from the perirhinal (PRh) cortex to the auditory cortex during learning and generalization of an auditory discrimination task. PRh input in the auditory cortex increased during learning and influenced performance in a categorization task. Chemogenetically silencing PRh input selectively disrupted the activity of auditory cortex layer 2/3 pyramidal neurons, and PRh photoactivation drove behavior toward overgeneralization. These findings highlight PRh input as a key modulatory pathway that shapes cortical dynamics underlying auditory learning and memory-guided sensory perception.

Pitch perception is fundamental to speech and music, yet the neural representation of pitch in auditory cortex remains poorly understood. We hypothesized that mice rely on temporal cues for pitch perception because of their wide auditory filters and high-frequency hearing. To investigate, we developed a mouse auditory filter bank model that estimates the temporal pitch salience of complex tones. We then trained mice to discriminate low vs. high fundamental frequencies (F0s) in complex tones and found that modeled temporal pitch salience was highly correlated with behavioral discrimination sensitivity. Widefield calcium imaging showed that the organization of periodotopy in auditory cortex predicts how well mice recognize temporal pitch cues. Mice trained on an F0 discrimination task had larger periodotopic maps with greater vector strengths than naïve mice, demonstrating experience-dependent expansion and refinement of temporal pitch maps in auditory cortex. Our findings establish mice as a robust model for studying the neural basis of temporal pitch perception.

Stimulus-specific adaptation is a key feature of sensory systems, where the repeated presentation of a stimulus results in diminishing neuronal responses specifically to that stimulus, but not to a different stimulus. Cortical neurons display varying degrees of stimulus-specific adaptation, with cortical circuits shaping the dynamics of these responses. Here, we use calcium imaging and optogenetic approaches to assess the contributions of SOM and PV neurons to stimulus-specific adaptation in the mouse auditory cortex. We find opposing and complementary effects of SOM and PV neurons on the adaptation of principal neurons which manifest as context-dependent SOM neuron-mediated enhancement and PV-neuron mediated suppression of responses that depend on the temporal repetition rate of the sounds. Our results suggest that SOM and PV neurons can engage in multiple operating regimes that exist in a continuum during sensory processing.

Harmonicity is a property of complex sounds such as vocalizations or music, but it remains unclear how harmonicity is processed in the auditory cortex (ACtx). Subregions of ACtx are thought to process harmonic stimuli differently. Selective responses to sound features in ACtx emerge hierarchically from primary ACtx (A1) L4 and secondary ACtx (A2) layer (L)2/3, which is believed to be the most responsive to harmonic sounds. Since harmonic stacks can range from 2 to >10 components, being more similar to naturalistic vocalizations, harmonic sensitivity might also arise hierarchically across layers and areas. We studied responses to harmonic stacks of 2–10 frequencies across A1 L4, A1 L2/3, and A2 L2/3 in adult male and female mice using in vivo two-photon microscopy. We found harmonic-sensitive neurons (HNs) responding only to harmonic stacks but not to individual frequencies in all areas at similar proportions. HNs showed highly nonlinear spectral integration of harmonic frequencies that decreased as the harmonic stacks became more complex. Specifically, onset-biased HNs showed greater nonlinearity than offset-biased HNs only in A1 L4. Moreover, HNs in A1 L4 exhibited higher signal correlation than A2 L2/3. Sound-responsive neurons in A1 L4 have the weakest noise correlation compared with A1 L2/3 and A2 L2/3. Together, harmonic sensitivity is not a unique feature of A2 L2/3 but is already established in A1 L4, where neurons robustly encode harmonic sounds through sparse connections.

Habituation is a crucial sensory filtering mechanism whose dysregulation can lead to a continuously intense world in disorders with hypersensitivity. Although habituation is often termed the simplest form of learning, its circuit mechanisms remain elusive. Conventional peripheral explanations fail to fully account for long-term habituation in complex sensory environments, leading to theories proposing top-down regulation. Here we evaluated two competing top-down mechanistic explanations for habituation in mice (growth in predictive filtering and waning of novelty-driven amplification) and identified an unexpected role for the orbitofrontal cortex (OFC) in predictive filtering. After daily sound exposure, neural habituation in the primary auditory cortex (A1) was reversed by inactivating the OFC. Top-down projections from the OFC, but not other frontal areas, carried predictive signals that grew with daily sound experience and suppressed A1 via somatostatin-expressing inhibitory neurons. Thus, prediction signals from the OFC cancel out anticipated stimuli by generating their 'negative images' in sensory cortices.

Introduction Spectral information of speech sounds is spatially arranged from low to high frequencies in the auditory cortex, known as tonotopic maps. Recently, we identified an envelope steepness map encoding the rise-ramp steepness of sounds, which is located orthogonally to the tonotopic map. Temporal components, particularly the sound envelope defined by the rise-ramp steepness, play a crucial role in speech perception. We hypothesized that the ability to sense temporal components of sounds, such as envelope steepness, would be diminished in aged mice. Methods Responses to variations in rise-ramp time were investigated using transcranial macroscale calcium imaging of the auditory cortices of GCaMP6f-expressing mice. Normalized distance relative to the rise-ramp time of 0.01 ms was plotted for each rise-ramp time (0.1, 1, 10, and 100 ms) in the logarithmic scale graph: the steeper the slope of the fitted regression lines, the greater the distance between the peaks of the rise-ramp time of 0.01 ms and 100 ms. The slope of this regression line was compared between the different age groups: 1/3 months and 6/9/12 months after birth. Results The slope of the fitted regression line for 5 kHz in the bilateral anterior auditory field and 20 kHz in the bilateral primary auditory field was significantly less steep in mice at 6/9/12 months after birth than at 1/3 months after birth. Conclusion The shorter distance from the peak of 0.01 ms to 100 ms in animals 6/9/12 months after birth suggests difficulty in the separation of the rise-ramp time in aged animals. These findings may support the cortical mechanisms underlying age-related decline in speech perception.

SignificanceAlthough the transmission of auditory information in the brain has been extensively studied, the mechanism underlying fine auditory discrimination remains incompletely understood. The basal region of the ventromedial nucleus of the thalamus (bVM) has recently been found to convey frequency-specific auditory information to the primary auditory cortex (A1). Inhibition of the bVM significantly impairs fine auditory discrimination in mice. These findings indicate that the bVM plays an important role in frequency information processing. However, direct functional evidence for tonotopic organization within the bVM is still lacking.AimWe aimed to investigate whether bVM neurons exhibit a spatially ordered frequency preference, using a simple yet efficient in vivo functional mapping strategy.ApproachTo characterize the response properties of bVM neurons projecting to A1, we combined Cre-dependent retrograde viral labeling with fiber photometry in awake mice. Using a “one recording site per animal” strategy, we systematically recorded from 26 distinct locations and successfully reconstructed the tonotopic map of the bVM.ResultsWe identified a mediolateral tonotopic gradient within the bVM, with best frequencies progressing from low in medial regions to high in lateral regions.ConclusionsOur findings provide direct functional evidence of tonotopic organization within the bVM, supporting its role as an auditory relay and its contribution to fine auditory discrimination.

Speech perception relies on two fundamental acoustic components: spectral and temporal. While spectral information is known to be represented in the auditory cortex through tonotopy, how temporal features are organized has remained unclear. Here, by varying onset rise-ramp steepness and frequencies, we reveal that temporal envelope steepness-a critical cue for phoneme discrimination and sound source perception-is systematically mapped in the mouse auditory cortex. Using widefield calcium imaging, we discovered that the envelope steepness is represented orthogonally to the tonotopic axis, forming a two-dimensional cortical map that mirrors the dual structure of sounds. This organization was observed in primary-like areas but not in higher-order-like areas, indicating distinct auditory processing streams. These findings uncover a principle of cortical organization, suggesting that the auditory cortex encodes sound along two independent axes and thereby provides a neural basis for the parallel processing of complex sounds such as speech and natural acoustic environments.

Previous studies based on layer specificity suggest that ascending signals from the thalamus to the sensory neocortex preserve spatially organized information, but it remains unknown whether sensory information descending from sensory neocortex to the thalamus also maintains such spatial organization pattern. By focusing on projection specificity, we mapped the tone response properties of two groups of cortical neurons in the primary auditory cortex (A1), based on the relationship between their specific connections to other regions and their function in ascending (thalamocortical recipient [TR] neurons) or descending (corticothalamic [CT] neurons) auditory information. A clear tonotopic gradient was observed among TR neurons, but not CT neurons. Additionally, CT neurons exhibited markedly higher heterogeneity in their frequency tuning and had broader bandwidth than TR neurons. These results reveal that the information flow descending from A1 to the thalamus via CT neurons is not arranged tonotopically, suggesting that the descending information flow possibly contributes to higher-order feedback processing of diverse auditory inputs.

Adolescence is a developmental period characterized by heightened plasticity. Yet, how ongoing development affects sensory processing and cognitive function is unclear. We investigated how adolescent (postnatal day 20–42) and adult (postnatal day 60–82) mice differ in performance on a pure tone Go/No-Go auditory discrimination task of varying difficulty. Using dense electrophysiological recordings, we measured spiking activity at single neuron resolution in the auditory cortex while mice were engaged in the task. As compared to adults, adolescent mice showed lower auditory discrimination performance in a difficult task. This difference in performance was due to higher response variability and weaker cognitive control expressed as higher lick bias. Adolescent and adult neuronal responses differed only slightly in representations of pure tones when measured outside the context of learning and the task. However, cortical representations after learning within the context of the task were markedly different. We found differences in stimulus- and choice-related activity at the single neuron level representations, as well as lower population-level decoding of the difficult task in adolescents. Overall, cortical decoding in adolescents was lower and slower, especially for difficult sound discrimination, reflecting immature cortical representations of sounds and choices. Notably, we found age-related differences, which were more pronounced after learning, reflecting the combined impact of age and learning. Our findings highlight distinct neurophysiological and behavioral profiles in adolescence, underscoring the ongoing development of cognitive control mechanisms and cortical plasticity during this sensitive developmental period.

Cross-modal responses are common in primary sensory cortex, yet a recent study showed that signals in primary visual cortex (V1) once labeled multisensory were instead more closely related to face movements. To what extent can multisensory processing in other modalities also be explained by face movements? We measured face movements and neural activity in awake mouse primary auditory cortex (A1) and V1 in response to visual and auditory stimulation as well as during spontaneous activity. We find that visual stimuli rarely evoke face movements, A1 responses to visual input remain robust even in the absence of face movement, and optogenetic silencing of V1 reduces A1 visual responses. Taken together, these results confirm that cross-modal responses in the auditory system are primarily sensory, not state driven, in origin.

The neural representations of acoustic features that differ in the location or timbre of the emitter elicit similar perceptions, suggesting the existence of a robust stimulus‐response function between complex sounds and the activity of neural populations at all stages of the auditory system. This hypothesis is tested by decoding a random sound stream, using spike trains from a biophysical model of the auditory nerve and from large‐scale recordings in the inferior colliculus, the auditory thalamus, and the auditory cortex of awake mice. At the level of individual neurons, the reliability of temporal and rate codes is found to decrease along the ascending auditory pathways. Rate coding is progressively favored with increasing independence of neuron frequency tuning. Firing in the auditory cortex is found to be synergistic, whereas that in subcortical areas is more redundant. Finally, combinatorial codes involving neural firing and neural silence within neuron pairs are shown to efficiently encode sound information, particularly in the auditory cortex. Overall, these findings reveal a progressive transformation of the neural code from an individual, redundant, and temporal code at the periphery to a more distributed rate‐based code in the auditory cortex.

Sound harmonicity is foundational in complex auditory stimuli like music and vocalizations but it remains unclear how such spectrally complex stimuli are processed in the auditory cortex (ACtx). Subregions of the auditory cortex process are thought to process harmonic stimuli differently, and secondary ACtx (A2) layer (L) 2/3 is believed to be the most selective. Selective responses to sound features in ACtx are thought to emerge hierarchically starting from A1 L4. Since the spectral complexity of harmonic stacks can range from two to more than ten components, harmonic selectivity and sensitivity might also arise hierarchically across layers and areas. We studied responses to simple and complex harmonic stacks across A1 L4, A1 L2/3, and A2 L2/3 in adult mice using in vivo two-photon microscopy. We found harmonic-sensitive neurons (HN) responding only to harmonic stacks but not to their pure-tone components in all areas at similar proportions. HNs showed non-linear processing of component tones with onset-responsive HNs showing greater nonlinearity, which decreased with harmonic complexity. Co-tuned HNs in A1 L4 exhibited the highest signal correlation, regardless of harmonic complexity. A1 L4 HNs also showed the lowest noise correlation with other neurons. Moreover, A1 L4 HNs achieve robust spectral integration and harmonic sensitivity by receiving diverse inputs and maintaining high signal correlation, ensuring independent, strong responses to harmonic stimuli. Therefore, harmonic sensitivity is present in A1 L4 and is not a unique feature of A2. Thus, tuning to complex spectral sounds is a fundamental property of ACtx and is already established in A1 L4. Significance statement Harmonics are essential in auditory perception, influencing how we process complex sounds like music and speech. This study reveals that neurons in the primary auditory cortex (A1) and secondary auditory cortex (A2) integrate simple and complex harmonic structures with distinct mechanisms of neuronal recruitment. A1 L4 harmonic-sensitive neurons (HNs) demonstrated strong, independent responses through high signal correlation and minimal noise correlation, suggesting a robust mechanism for spectral integration. Our results show that harmonic relationships are already extracted at the input layers of A1, and that HNs show non-linear facilitative integration. Thus, tuning to sounds of complex spectral contents might be a fundamental processing function of the auditory cortex and is already established in A1 L4, which receives major thalamic inputs.

The insular cortex is involved in a wide range of auditory functions in the mammalian brain. We studied the organization and basic response properties of auditory neurons in the insular cortex and adjacent areas by recording responses to sound stimuli in anesthetized female rats. Auditory neurons were present in an insulo-parietal auditory field that spans the boundary between the posterior insula, particularly in the granular insular cortex, and the ventral part of the adjacent nonprimary somatosensory fields. Confirming previous reports, neurons in this field had narrow tuning and were preferentially tuned to relatively low frequencies (<16 kHz). Intriguingly, auditory units in this field displayed shorter minimal latencies than units in the primary auditory cortex, as well as weaker sensitivity to deviance. These results establish the existence of a rapid auditory information stream with distinct properties through the insulo-parietal cortex that may parallel the pathway through the primary and anterior auditory fields.

Auditory processing in the cerebral cortex is considered to begin with thalamocortical inputs to layer 4 (L4) of the primary auditory cortex (A1). In this canonical model, A1 L4 inputs initiate a hierarchical cascade that propagates to higher-order cortices for slower integration of complex sounds. Here, we identify parallel ascending pathways in mice that bypass A1 and directly reach the secondary auditory cortex (A2), alongside the canonical hierarchical route. We found that layer 6 (L6) of both A1 and A2 receive short-latency (<10 ms) sound inputs via higher-order thalamic nuclei. Additionally, A2 L4 is innervated by a caudal subdivision of the traditionally defined primary thalamus, which we now re-classify as non-primary. Notably, both identified thalamic regions receive projections from distinct subdivisions of the higher-order inferior colliculus, which in turn receive direct projections from cochlear nucleus neurons. Thus, higher-order auditory cortex integrates both slower, pre-processed information and rapid, direct sensory inputs, enabling parallel processing of fast sensory information across cortical areas.

Analyzing the functional connectivity of the brain is an enormous challenge, as deciphering functional connectivity requires knowledge of functional responses and connections. One promising strategy is analyzing the spatial pattern of activity correlations across cell populations. In the primary auditory cortex (A1), cells respond to different sound features. On the large scale, there exists a tonotopic map, which is fractured at the small scale, raising the question of whether functional connections are spatially ordered or disordered. To test whether functional connectivity on a local and a global scale is also disordered, we first designed a robust statistical model to estimate parameters and test for the significance of the estimated correlation maps. We developed an inference method that allows efficient model fitting and statistical testing to project the correlation maps to 2D space. We then performed in vivo two-photon calcium imaging in layer 2/3 of A1 with pure tones (PT) or a combination of two tones (TT; harmonically related or not). We found that the spatial patterns of signal correlations (SCs) depend on the type of sound stimuli that were presented. The functional 2D maps of PT-driven SCs are more restricted to local neurons than TT signal correlations which showed more global textures. 2D SC patterns for harmonic stimuli showed spatially distinct relationships. TT SCs revealed spatially precise functional connectivity between harmonically related neurons. Thus, even though the frequency preference of neighboring neurons in A1 is functionally diverse, the functional connection pattern of these neurons is functionally precise and harmonically related.

Negative feedback of the cochlear efferent system plays a critical role in control of hearing sensitivity and protection from noise trauma. Type II auditory nerves (ANs) innervate outer hair cells (OHCs) in the cochlea and provide an input to the cochlear efferent system to achieve hearing sensitivity controlling and protection; in particular, medial olivocochlear efferent nerves innervate OHCs to control OHC electromotility, which is an active cochlear amplifier in mammals. However, little is known about channel information underlying type II AN activity and consequent function. Here, we report that ATP-gated P2x7 receptor had a predominant expression at type II spiral ganglion (SG) neurons and the synaptic areas under inner hair cells and OHCs with lateral and medial olivocochlear efferent nerves. Knockout (KO) of P2x7 increased hearing sensitivity with enhanced acoustic startle response, auditory brainstem response, and cochlear microphonics by increasing OHC electromotility. P2x7 KO also increased susceptibility to noise and exacerbated ribbon synapse degeneration. Middle-level noise exposure could impair active cochlear mechanics resulting in hearing loss in P2x7 KO mice. These data demonstrate that P2x7 receptors have a critical role in type II SG neuron’s function and the cochlear efferent system to control hearing sensitivity; deficiency of P2x7 receptors can impair type II SG neuron’s function and the cochlear efferent suppression leading to increase of active cochlear amplification and hearing oversensitivity, i.e., hyperacusis, and susceptibility to noise, which may also associate with other hearing disorders, such as tinnitus.

Socially effective vocal communication requires brain regions that encode expressive and receptive aspects of vocal communication in a social context-dependent manner. Here, we combined a novel behavioral assay with microendoscopic calcium imaging to interrogate neuronal activity (regions of interest [ROIs]) in the posterior insula (pIns) in socially interacting mice as they switched rapidly between states of vocal expression and reception. We found that largely distinct subsets of pIns ROIs were active during vocal expression and reception. Notably, pIns activity during vocal expression increased prior to vocal onset and was also detected in congenitally deaf mice, pointing to a motor signal. Furthermore, receptive pIns activity was modulated strongly by social context. Lastly, tracing experiments reveal that deep-layer neurons in the pIns directly bridge the auditory thalamus to a midbrain vocal gating region. Therefore, the pIns is a site that encodes vocal expression and reception in a manner that depends on social context.