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.

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.

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.

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.

The thalamocortical pathways exhibit neuroplasticity not only during the critical period but also in adulthood. In this study, we investigated how cholecystokinin (CCK) modulates age-dependent thalamocortical plasticity. Our findings demonstrated that CCK is expressed in thalamocortical neurons and that high-frequency stimulation (HFS) of the thalamocortical pathway triggers the release of CCK in auditory cortex (ACx), as detected by a CCK sensor. HFS of the medial geniculate body (MGB) induced thalamocortical long-term potentiation (LTP) in wild-type young adult mice. However, knockdown of CCK expression in MGB neurons or blockade of the CCK-B receptor (CCKBR) in the ACx abolished HFS-induced LTP. Interestingly, this LTP could not be elicited in juvenile (3-week-old) or aged mice (over 18-month-old) due to distinct mechanisms: the absence of CCKBR in juveniles and the inability to release CCK in aged mice. Notably, exogenous administration of CCK into the ACx rescued LTP in aged mice and significantly improved frequency discrimination. These findings highlight the potential of CCK as a therapeutic intervention for ameliorating neuroplasticity deficits associated with thalamocortical connectivity.

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.

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.

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.

Lateralized auditory processing is essential for specialized functions such as speech processing, typically dominated by the Left Auditory Cortex (ACx) in humans. Hemispheric specializations also occur in the adult mouse ACx, but their developmental origins are unclear. Our study finds that the Left and Right ACx in mice reach developmental milestones at different ages. Thalamocortical responses and maturation of synaptic dynamics develop earlier in the Right ACx than the Left. We show that this timing offset predicts hemisphere-dependent differences in sensory-driven plasticity. Juvenile tone exposure at specific times results in imbalanced adult tone frequency representations in the Right and Left ACx. Additionally, sex influences the timing of plasticity; female Right ACx plasticity occurs before male Right ACx, and female Left ACx aligns with male Right ACx plasticity. Our findings demonstrate that sex and hemispheric identity drive asynchronous development and contribute to functional differences in sensory cortices.

While evolutionary cortical expansion is thought to underlie the evolution of human cognitive capabilities, excessive developmental expansion can lead to megalencephaly, often found in neurodevelopmental disorders. Still, little is known about how the overproduction of cortical neurons during development affects cortical processing and behavior in later life. Here we show that developmental overproduction of cortical superficial neurons impairs auditory processing in adult mice. We applied XAV939 to overproduce cortical superficial excitatory neurons during development. XAV939-treated adult mice exhibited auditory behavioral deficits and abnormal auditory cortical processing. Furthermore, we found fewer functional monosynaptic connections between cortical putative excitatory neurons. Altogether, our results suggest that abnormal auditory cortical processing contributes to the atypical auditory detectability in XAV939-treated mice. Although the expansion of cortical size is evolutionarily advantageous, an abnormal expansion during development can result in detrimental effects on cortical processing and perceptual behavior in adulthood.

Transgenic mice provide unprecedented access to manipulate and visualize neural circuits; however, those on a C57BL/6 background develop progressive hearing loss, significantly confounding systems-level and behavioral analysis. While outbreeding can limit hearing loss, it introduces strain variability and complicates the generation of complex genotypes. Here, we propose an approach to preserve hearing by crossing transgenic mice with congenic B6.CAST- Cdh23 Ahl + mice, which maintain low-threshold hearing into adulthood. Widefield and two-photon imaging of the auditory cortex revealed that 2.5-month-old C57BL/6 mice exhibit elevated thresholds to high-frequency tones and widespread cortical reorganization, with most neurons responding best to lower frequencies. In contrast, Ahl+ C57BL/6 mice exhibited robust neural responses across tested frequencies and sound levels (4–64 kHz, 30–90 dB SPL) and retained low thresholds into adulthood. Our approach offers a cost-effective solution for generating complex genotypes and facilitates more interpretable systems neuroscience research by eliminating confounding effects from hearing loss.

Sound location coding has been extensively studied at the central nucleus of the mammalian inferior colliculus (CNIC), supporting a population code. However, this population code has not been extensively characterized on the single-trial level with simultaneous recordings or at other anatomical regions like the dorsal cortex of inferior colliculus (DCIC), which is relevant for learning-induced experience dependent plasticity. To address these knowledge gaps, here we made in two complementary ways large-scale recordings of DCIC populations from awake mice in response to sounds delivered from 13 different frontal horizontal locations (azimuths): volumetric two-photon calcium imaging with ~700 cells simultaneously recorded at a relatively low temporal resolution, and high-density single-unit extracellular recordings with ~20 cells simultaneously recorded at a high temporal resolution. Independent of the method, the recorded DCIC population responses revealed substantial trial-to-trial variation (neuronal noise) which was significantly correlated across pairs of neurons (noise correlations) in the passively listening condition. Nevertheless, decoding analysis supported that these noisy response patterns encode sound location on the single-trial basis, reaching errors that match the discrimination ability of mice. The detected noise correlations contributed to minimize the error of the DCIC population code of sound azimuth. Altogether these findings point out that DCIC can encode sound location in a similar format to what has been proposed for CNIC, opening exciting questions about how noise correlations could shape this code in the context of cortico-collicular input and experience-dependent plasticity.

Recent studies have shown promising therapeutic potential for psychedelics in treating neuropsychiatric conditions. Musical experience during psilocybin-assisted therapy is predictive of treatment outcome, yet little is known about how psilocybin affects auditory processing. Here, we conducted two-photon imaging experiments in auditory cortex of awake mice that received a dose of psilocybin. Our results suggest that psilocybin modulates the roles of intrinsic neural activity versus stimulus-driven influences on auditory perception.

Salicylate is commonly used to induce tinnitus in animals, but its underlying mechanism of action is still debated. We therefore tested its effects on the firing properties of neurons in the mouse inferior colliculus (IC). Salicylate induced a large decrease in the spontaneous activity and an increase of ∼20 dB SPL in the minimum threshold of single units. In response to sinusoidally modulated noise (SAM noise) single units showed both an increase in phase locking and improved rate coding. Mice also became better at detecting amplitude modulations, and a simple threshold model based on the IC population response could reproduce this improvement. The responses to dynamic random chords (DRCs) suggested that the improved AM encoding was due to a linearization of the cochlear output, resulting in larger contrasts during SAM noise. These effects of salicylate are not consistent with the presence of tinnitus, but should be taken into account when studying hyperacusis.

Changes in sound-evoked responses in the auditory cortex (ACtx) occur during learning, but how learning alters neural responses in different ACtx subregions and changes their interactions is unclear. To address these questions, we developed an automated training and widefield imaging system to longitudinally track the neural activity of all mouse ACtx subregions during a tone discrimination task. We find that responses in primary ACtx are highly informative of learned stimuli and behavioral outcomes throughout training. In contrast, representations of behavioral outcomes in the dorsal posterior auditory field, learned stimuli in the dorsal anterior auditory field, and inter-regional correlations between primary and higher-order areas are enhanced with training. Moreover, ACtx response changes vary between stimuli, and such differences display lag synchronization with the learning rate. These results indicate that learning alters functional connections between ACtx subregions, inducing region-specific modulations by propagating behavioral information from primary to higher-order areas.

Autism spectrum disorder (ASD) is often associated with social communication impairments and specific sound processing deficits, for example, problems in following speech in noisy environments. To investigate underlying neuronal processing defects located in the auditory cortex (AC), we performed two-photon Ca 2+ imaging in FMR1 ( fragile X messenger ribonucleoprotein 1 ) knock-out (KO) mice, a model for fragile X syndrome (FXS), the most common cause of hereditary ASD in humans. For primary AC (A1) and the anterior auditory field (AAF), topographic frequency representation was less ordered compared with control animals. We additionally analyzed ensemble AC activity in response to various sounds and found subfield-specific differences. In A1, ensemble correlations were lower in general, while in secondary AC (A2), correlations were higher in response to complex sounds, but not to pure tones. Furthermore, sound specificity of ensemble activity was decreased in AAF. Repeating these experiments 1 week later revealed no major differences regarding representational drift. Nevertheless, we found subfield- and genotype-specific changes in ensemble correlation values between the two times points, hinting at alterations in network stability in FMR1 KO mice. These detailed insights into AC network activity and topography in FMR1 KO mice add to the understanding of auditory processing defects in FXS.

Psilocybin is a serotonergic psychedelic believed to have therapeutic potential for neuropsychiatric conditions. Despite well-documented prevalence of perceptual alterations, hallucinations, and synesthesia associated with psychedelic experiences, little is known about how psilocybin affects sensory cortex or alters the activity of neurons in awake animals. To investigate, we conducted 2-photon imaging experiments in auditory cortex of awake mice and video analysis of mouse behavior, both at baseline and during psilocybin treatment. We found biphasic effects of psilocybin on behavioral and cortical activity. A 2 mg/kg dose of psilocybin initially increased behavioral activity and neural responses to sound. 30 minutes post-dose, mice became behaviorally hypoactive and cortical responses to sound decreased, while neural response variance and noise correlations increased. In contrast, neuronal selectivity for auditory stimuli remained stable during psilocybin treatment. Our results suggest that psilocybin modulates the role of intrinsic versus stimulus-driven activity in sensory cortex, while preserving fundamental sensory processing. Graphical Abstract. Summary of psilocybin’s effect on auditory cortical responses to sound in mice. 30 minutes after injecting the inert vehicle saline, typical auditory responses occur within a relatively narrow range of possible amplitudes, i.e., each neuron’s response variance is weakly correlated with a neighboring neuron’s response variance. In contrast, 30 minutes after injecting 2 mg/kg of psilocybin, population response variance becomes more correlated between individual neurons, and the range of response amplitudes increases. These findings suggest that psilocybin modulates the role of intrinsic versus stimulus-driven neural activity in sensory perception, which may serve as a basis for auditory hallucination at the level of neuronal micro-circuits.

The sensory neocortex has been suggested to be a substrate for long-term memory storage, yet which exact single cells could be specific candidates underlying such long-term memory storage remained neither known nor visible for over a century. Here, using a combination of day-by-day two-photon Ca 2+ imaging and targeted single-cell loose-patch recording in an auditory associative learning paradigm with composite sounds in male mice, we reveal sparsely distributed neurons in layer 2/3 of auditory cortex emerged step-wise from quiescence into bursting mode, which then invariably expressed holistic information of the learned composite sounds, referred to as holistic bursting (HB) cells. Notably, it was not shuffled populations but the same sparse HB cells that embodied the behavioral relevance of the learned composite sounds, pinpointing HB cells as physiologically-defined single-cell candidates of an engram underlying long-term memory storage in auditory cortex.