Electrophysiology publications

TRPA1 channels are expressed in nociceptive neurons, where they detect noxious stimuli, and in the mammalian cochlea, where their function is unknown. Here we show that TRPA1 activation in the supporting non-sensory Hensen’s cells of the mouse cochlea causes prolonged Ca 2+ responses, which propagate across the organ of Corti and cause long-lasting contractions of pillar and Deiters’ cells. Caged Ca 2+ experiments demonstrated that, similar to Deiters’ cells, pillar cells also possess Ca 2+ -dependent contractile machinery. TRPA1 channels are activated by endogenous products of oxidative stress and extracellular ATP. Since both these stimuli are present in vivo after acoustic trauma, TRPA1 activation after noise may affect cochlear sensitivity through supporting cell contractions. Consistently, TRPA1 deficiency results in larger but less prolonged noise-induced temporary shift of hearing thresholds, accompanied by permanent changes of latency of the auditory brainstem responses. We conclude that TRPA1 contributes to the regulation of cochlear sensitivity after acoustic trauma.

Intrinsically generated neural activity propagates through the developing auditory system to promote maturation and refinement of sound processing circuits prior to hearing onset. This early patterned activity is induced by non-sensory supporting cells in the organ of Corti, which are highly interconnected through gap junctions containing connexin 26 ( Gjb2 ). Although loss of function mutations in Gjb2 impair cochlear development and are the most common cause of congenital deafness, it is not known if these variants disrupt spontaneous activity and the developmental trajectory of sound processing circuits in the brain. Here, we show in a new mouse model of Gjb2- mediated congenital deafness that cochlear supporting cells adjacent to inner hair cells (IHCs) unexpectedly retain intercellular coupling and the capacity to generate spontaneous activity, exhibiting only modest deficits prior to hearing onset. Supporting cells lacking Gjb2 elicited coordinated activation of IHCs, leading to coincident bursts of activity in central auditory neurons that will later process similar frequencies of sound. Despite alterations in the structure of the sensory epithelium, hair cells within the cochlea of Gjb2 -deficient mice were intact and central auditory neurons could be activated within appropriate tonotopic domains by loud sounds at hearing onset, indicating that early maturation and refinement of auditory circuits was preserved. Only after cessation of spontaneous activity following hearing onset did progressive hair cell degeneration and enhanced auditory neuron excitability manifest. This preservation of cochlear spontaneous neural activity in the absence of connexin 26 may increase the effectiveness of early therapeutic interventions to restore hearing.

Synaptic zinc is a neuromodulator that shapes synaptic transmission and sensory processing. The maintenance of synaptic zinc is dependent on the vesicular zinc transporter, ZnT3. Hence, the ZnT3 knockout mouse has been a key tool for studying the mechanisms and functions of synaptic zinc. However, the use of this constitutive knockout mouse has notable limitations, including developmental, compensatory, and brain and cell type specificity issues. To overcome these limitations, we developed and characterized a dual recombinase transgenic mouse, which combines the Cre and Dre recombinase systems. This mouse allows for tamoxifen-inducible Cre-dependent expression of exogenous genes or knockout of floxed genes in ZnT3-expressing neurons and DreO-dependent region and cell type–specific conditional ZnT3 knockout in adult mice. Using this system, we reveal a neuromodulatory mechanism whereby zinc release from thalamic neurons modulates N -methyl- d -aspartate receptor activity in layer 5 pyramidal tract neurons, unmasking previously unknown features of cortical neuromodulation.

Adenylyl cyclase 6 (AC6) synthesizes second messenger cAMP in G protein-coupled receptor (GPCR) signaling. In cochlear hair cells, AC6 distribution relies on an adhesion GPCR, ADGRV1, which is associated with Usher syndrome (USH), a condition of combined hearing and vision loss. ADGRV1 is a component of the USH type 2 (USH2) protein complex in hair cells and photoreceptors. However, the role of AC6 in the inner ear and retina has not been explored. Here, we found that AC6 distribution in hair cells depends on the USH2 protein complex integrity. Several known AC6 regulators and effectors, which were previously reported to participate in ADGRV1 signaling in vitro, are localized to the stereociliary compartments that overlap with AC6 distribution in hair cells. In young AC6 knockout ( Adcy6 −/− ) mice, the activity of cAMP-dependent protein kinase, but not Akt kinase, is altered in cochleas, while both kinases are normal in vestibular organs. Adult Adcy6 −/− mice however exhibit normal hearing function. AC6 is expressed in mouse retinas but rarely in photoreceptors. Adcy6 −/− mice have slightly enhanced photopic but normal scotopic vision. Therefore, AC6 may participate in the ADGRV1 signaling in hair cells but AC6 is not essential for cochlear and retinal development and maintenance.

Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations in vivo. Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca2+ imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca2+ imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.

Quantitative and mechanistic understanding of learning and long-term memory at the level of single neurons in living brains require highly demanding techniques. A specific need is to precisely label one cell whose firing output property is pinpointed amidst a functionally characterized large population of neurons through the learning process and then investigate the distribution and properties of dendritic inputs. Here, we disseminate an integrated method of daily two-photon neuronal population Ca 2+ imaging through an auditory associative learning course, followed by targeted single-cell loose-patch recording and electroporation of plasmid for enhanced chronic Ca 2+ imaging of dendritic spines in the targeted cell. Our method provides a unique solution to the demand, opening a solid path toward the hard-cores of how learning and long-term memory are physiologically carried out at the level of single neurons and synapses.

Cochlear implants are well established to treat severe hearing impairments. Despite many different approaches to reduce the formation of connective tissue after electrode insertion and to keep electrical impedances low, results are not yet satisfying. Therefore, the aim of the current study was to combine the incorporation of 5% dexamethasone in the silicone body of the electrode array with an additional polymeric coating releasing diclofenac or the immunophilin inhibitor MM284, some anti-inflammatory substances not yet tested in the inner ear. Guinea pigs were implanted for four weeks and hearing thresholds were determined before implantation and after the observation time. Impedances were monitored over time and, finally, connective tissue and the survival of spiral ganglion neurons (SGNs) were quantified. Impedances increased in all groups to a similar extent but this increase was delayed in the groups with an additional release of diclofenac or MM284. Using Poly-L-lactide (PLLA)-coated electrodes, the damage caused during insertion was much higher than without the coating. Only in these groups, connective tissue could extend to the apex of the cochlea. Despite this, numbers of SGNs were only reduced in PLLA and PLLA plus diclofenac groups. Even though the polymeric coating was not flexible enough, MM284 seems to especially have potential for further evaluation in connection with cochlear implantation.

We examined the effect of neonatal deafening on frequency-specific pathways for processing of interaural time differences (ITDs) in cochlear-implant stimuli. Animal studies have demonstrated differences in neural ITD sensitivity in the inferior colliculus (IC) depending on the intracochlear location of intracochlear stimulating electrodes. We used neonatally deafened (ND) rats of both sexes and recorded the responses of single neurons in the IC to electrical stimuli with ITDs delivered to the apical or basal cochlea and compared them with acutely deafened (AD) rats of both sexes with normal hearing (NH) during development. We found that neonatal deafness significantly impacted the ITD sensitivity and the ITD tuning patterns restricted to apically driven IC neurons. In ND rats, the ITD sensitivity of apically driven neurons is reduced to values similar to basally driven neurons. The prevalence of ITD-sensitive apical neurons with a peak-shaped ITD tuning curve, which may reflect predominant input from the medial superior olivary (MSO) complex, in ND rats was diminished compared with that in AD rats (67%, AD vs 40%, ND). Conversely, monotonic-type responses rarely occurred in AD rats (14%) but were approximately equally as prevalent as peak-type tuning curves in ND rats (42%). Nevertheless, in ND rats, the ITD at the maximum slope of the ITD tuning curve was still more concentrated within the physiological ITD range in apically driven than in basally driven neurons. These results indicate that the development of high ITD sensitivity processed by low-frequency pathways depends on normal auditory experience and associated biases in ITD tuning strategies.

From myriads of ongoing stimuli, the brain creates a fused percept of the environment. This process, which culminates in perceptual binding, is presumed to occur through the operations of multisensory neurons that occur throughout the brain. However, because different brain areas receive different inputs and have different cytoarchitechtonics, it would be expected that local multisensory features would also vary across regions. The present study investigated that hypothesis using multiple single-unit recordings from anesthetized cats in response to controlled, electronically-generated separate and combined auditory, visual, and somatosensory stimulation. These results were used to compare the multisensory features of neurons in cat primary auditory cortex (A1) with those identified in the nearby higher-order auditory region, the Dorsal Zone (DZ). Both regions exhibited the same forms of multisensory neurons, albeit in different proportions. Multisensory neurons exhibiting excitatory or inhibitory properties occurred in similar proportions in both areas. Also, multisensory neurons in both areas expressed similar levels of multisensory integration. Because responses to auditory cues alone were so similar to those that included non-auditory stimuli, it is proposed that this effect represents a mechanism by which multisensory neurons subserve the process of perceptual binding.

Progressive functional deterioration in the cochlea is associated with age-related hearing loss (ARHL). However, the cellular and molecular basis underlying cochlear aging remains largely unknown. Here, we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging, in which we characterized aging-related transcriptomic changes in 27 different cochlear cell types across five different time points. Overall, our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging, highlights unexpected age-related transcriptional fluctuations in intermediate cells localized in the stria vascularis (SV) and demonstrates that upregulation of endoplasmic reticulum (ER) chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging. Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related SV atrophy and hence delay the progression of ARHL.

Understanding functional correlations between the activities of neuron populations is vital for the analysis of neuronal networks. Analyzing large‐scale neuroimaging data obtained from hundreds of neurons simultaneously poses significant visualization challenges. We developed V‐NeuroStack, a novel network visualization tool to visualize data obtained using calcium imaging of spontaneous activity of neurons in a mouse brain slice as well as in vivo using two‐photon imaging. V‐NeuroStack creates 3D time stacks by stacking 2D time frames for a time‐series dataset. It provides a web interface to explore and analyze data using both 3D and 2D visualization techniques. Previous attempts to analyze such data have been limited by the tools available to visualize large numbers of correlated activity traces. V‐NeuroStack's 3D view is used to explore patterns in dynamic large‐scale correlations between neurons over time. The 2D view is used to examine any timestep of interest in greater detail. Furthermore, a dual‐line graph provides the ability to explore the raw and first‐derivative values of activity from an individual or a functional cluster of neurons. V‐NeuroStack can scale to datasets with at least a few thousand temporal snapshots. It can potentially support future advancements in in vitro and in vivo data capturing techniques to bring forth novel hypotheses by allowing unambiguous visualization of massive patterns in neuronal activity data.

Nocturnal moths evolved ultrasound-triggered escape maneuvers for avoiding predatory bats emitting ultrasonic echolocation calls. Using ultrasound for pest control is not a novel concept, but the technique has not been systemized because of the moths’ habituation to sounds and the narrow directionality of conventional ultrasound speakers. Here, we report the use of pulsed ultrasonic white noise, which contributes to achieving ecologically concordant plant protection. An ultrasonic pulse, which is temporal mimicry of the search-phase pulse in the echolocation calls of a sympatric bat, was identified using neuroethological screening of eared moth–repelling ultrasounds; these pulses elicit flight-stopping reactions in moths but have no or little auditory adaptation. Such repellent ultrasounds broadcast from the cylindrical omni-azimuth ultrasound emitters suppressed the intrusion of gravid females of pest moths into cultivation fields. Thus, egg numbers and plant damage by hatched larvae were drastically reduced, enabling farmers to substantially skip applications of chemical insecticides for controlling moth pests.

Vertigo is a debilitating disease affecting 15–20% of adults worldwide. Vestibular peripheral vertigo is the most common cause of vertigo, often due to Meniere's disease and benign paroxysmal positional vertigo. Although some vertigo symptoms can be controlled by conservative treatment and/or vestibular rehabilitation therapy, these treatments do not work for some patients. Semicircular canal occlusion surgery has proven to be very effective for these patients with intractable vertigo. However, its application is limited due to concern that the procedure will disrupt normal hearing. In this study, we investigated if occlusion of two semicircular canals would jeopardize auditory function by comparing auditory function and hair cell morphology between the surgical and contralateral ears before and after the surgery in a mouse model. By measuring the auditory brainstem response and distortion product otoacoustic emission 4 weeks post-surgery, we show that auditory function does not significantly change between the surgical and contralateral ears. In addition, confocal imaging has shown no hair cell loss in the cochlear and vestibular sensory epithelia, and scanning electron microscopy also indicates normal stereocilia morphology in the surgical ear. More importantly, the endocochlear potential measured from the surgical ear is not significantly different than that seen in the contralateral ear. Our study suggests that occlusion of two semicircular canals does not disrupt normal hearing in the mouse model, providing a basis to extend the procedure to patients, even those with normal hearing, benefitting more patients with intractable vertigo attacks.

Tinnitus is the phantom perception of sound that has no external source. A neurological signature of tinnitus, and the frequently associated hyperacusis, is an imbalance between excitatory and inhibitory activity in the central auditory system (CAS), leading to dysregulated network excitability. The large conductance, calcium-activated potassium (BK) channel is a key player in pre- and post-synaptic excitability through its mediation of K + currents. Changes in BK channel activity are associated with aberrant network activity in sensory regions of the CNS, raising the possibility that BK channel modulation could regulate activity associated with tinnitus and hyperacusis. To test whether BK channel openers are able to suppress biomarkers of drug-induced tinnitus and hyperacusis, the 1,3,4 oxadiazole BMS-191011 was given to young adult CBA mice that had been administered 250 mg/kg sodium salicylate (SS). Systemic treatment with BMS-191011 reduced behavioral manifestations of SS-induced tinnitus, but not hyperacusis, probed via the gap-in-noise startle response method. Systemic BMS-191011 treatment did not influence SS-induced increases in auditory brainstem response functions, but local application at the inferior colliculus did reverse SS-suppressed spontaneous activity, particularly in the frequency region of the tinnitus percept. Thus, action of BMS-191011 in the inferior colliculus may contribute to the reduction in behaviorally measured tinnitus. Together, these findings support the utility of BK channel openers in reducing central auditory processing changes associated with the formation of the tinnitus percept.

Sensory responses typically vary depending on the recent history of sensory experience. This is essential for processes, including adaptation, efficient coding, and change detection. In the auditory cortex (AC), the short-term history dependence of sound-evoked (onset) responses has been well characterized. Yet many AC neurons also respond to sound terminations, and little is known about the history dependence of these "offset" responses, whether the short-term dynamics of onset and offset responses are correlated, or how these properties are distributed among cell types. Here we presented awake male and female mice with repeating noise burst stimuli while recording single-unit activity from primary AC. We identified parvalbumin and somatostatin interneurons through optotagging, and also separated narrow-spiking from broad-spiking units. We found that offset responses are typically less depressive than onset responses, and this result was robust to a variety of stimulus parameters, controls, measurement types, and selection criteria. Whether a cell's onset response facilitates or depresses does not predict whether its offset response facilitates or depresses. Cell types differed in the dynamics of their onset responses, and in the prevalence, but not the dynamics, of their offset responses. Finally, we clustered cells according to spiking responses and found that response clusters were associated with cell type. Each cluster contained cells of several types, but even within a cluster, cells often showed cell type-specific response dynamics. We conclude that onset and offset responses are differentially influenced by recent sound history, and discuss the implications of this for the encoding of ongoing sound stimuli.SIGNIFICANCE STATEMENT Sensory neuron responses depend on stimulus history. This history dependence is crucial for sensory processing, is precisely controlled at individual synapses and circuits, and is adaptive to the specific requirements of different sensory systems. In the auditory cortex, neurons respond to sound cessation as well as to sound itself, but how history dependence is used along this separate, "offset" information stream is unknown. We show that offset responses are more facilitatory than sound responses, even in neurons where sound responses depress. In contrast to sound onset responses, offset responses are absent in many cells, are relatively homogeneous, and show no cell type-specific differences in history dependence. Offset responses thus show unique response dynamics, suggesting their unique functions.

In everyday behavior, sensory systems are in constant competition for attentional resources, but the cellular and circuit-level mechanisms of modality-selective attention remain largely uninvestigated. We conducted translaminar recordings in mouse auditory cortex (AC) during an audiovisual (AV) attention shifting task. Attending to sound elements in an AV stream reduced both pre-stimulus and stimulus-evoked spiking activity, primarily in deep-layer neurons and neurons without spectrotemporal tuning. Despite reduced spiking, stimulus decoder accuracy was preserved, suggesting improved sound encoding efficiency. Similarly, task-irrelevant mapping stimuli during inter-trial intervals evoked fewer spikes without impairing stimulus encoding, indicating that attentional modulation generalized beyond training stimuli. Importantly, spiking reductions predicted trial-to-trial behavioral accuracy during auditory attention, but not visual attention. Together, these findings suggest auditory attention facilitates sound discrimination by filtering sound-irrelevant background activity in AC, and that the deepest cortical layers serve as a hub for integrating extramodal contextual information.

We introduce two EEG techniques, one based on conventional monopolar electrodes and one based on a novel tripolar electrode, to record for the first time auditory brainstem responses (ABRs) from the scalp of unanesthetized, unrestrained big brown bats. Stimuli were frequency-modulated (FM) sweeps varying in sweep direction, sweep duration, and harmonic structure. As expected from previous invasive ABR recordings, upward-sweeping FM signals evoked larger amplitude responses (peak-to-trough amplitude in the latency range of 3–5 ms post-stimulus onset) than downward-sweeping FM signals. Scalp-recorded responses displayed amplitude-latency trading effects as expected from invasive recordings. These two findings validate the reliability of our noninvasive recording techniques. The feasibility of recording noninvasively in unanesthetized, unrestrained bats will energize future research uncovering electrophysiological signatures of perceptual and cognitive processing of biosonar signals in these animals, and allows for better comparison with ABR data from echolocating cetaceans, where invasive experiments are heavily restricted.

Urethane has little effect on nervous system and is often used in neuroscience studies. However, the effect of urethane in neurons is not thoroughly clear. In this study, we investigated changes in neuron responses to tones in inferior colliculus during urethane anesthesia. As urethane was metabolized, the best and characteristic frequencies did not obviously change, but the minimal threshold (MT) remained relatively stable or was elevated. The frequency tuning bandwidth at 60 dB SPL (BW 60dBSPL ) remained unchanged or decreased, and the average evoked spike of effective frequencies at 60 dB SPL (ES 60dBSPL ) gradually decreased. Although the average evoked spike of effective frequencies at a tone intensity of 20 dB SPL above MT (ES 20dBSPLaboveMT ) decreased, the frequency tuning bandwidth at a tone intensity of 20 dB SPL above MT (BW 20dBSPLaboveMT ) did not change. In addition, the changes in MT, ES 60dBSPL , BW 60dBSPL , and ES 20dBSPLaboveMT increased with the MT in pre-anesthesia awake state (MT pre−anesthesiaawake ). In some neurons, the MT was lower, BW 60dBSPL was broader, and ES 60dBSPL and ES 20dBSPLaboveMT were higher in urethane anesthesia state than in pre-anesthesia awake state. During anesthesia, the inhibitory effect of urethane reduced the ES 20dBSPLaboveMT , but did not change the MT, characteristic frequency, or BW 20dBSPLaboveMT . In the recording session with the strongest neuron response, the first spike latency did not decrease, and the spontaneous spike did not increase. Therefore, we conclude that urethane can reduce/not change the MT, increase the evoked spike, or broaden/not change the frequency tuning range, and eventually improve the response of auditory neurons to tone with or without “pushing down” the tonal receptive field in thresholding model. The improved effect increases with the MT pre−anesthesiaawake of neurons. The changes induced by the inhibitory and improved effects of urethane abide by similar regularities, but the change directions are contrary. The improvement mechanism may be likely due to the increase in the ratio of excitatory/inhibitory postsynaptic inputs to neurons.

Information flow in the sensory cortex has been described as a predominantly feedforward sequence with deep layers as the output structure. Although recurrent excitatory projections from layer 5 (L5) to superficial L2/3 have been identified by anatomical and physiological studies, their functional impact on sensory processing remains unclear. Here, we use layer-selective optogenetic manipulations in the primary auditory cortex to demonstrate that feedback inputs from L5 suppress the activity of superficial layers regardless of the arousal level, contrary to the prediction from their excitatory connectivity. This suppressive effect is predominantly mediated by translaminar circuitry through intratelencephalic neurons, with an additional contribution of subcortical projections by pyramidal tract neurons. Furthermore, L5 activation sharpened tone-evoked responses of superficial layers in both frequency and time domains, indicating its impact on cortical spectro-temporal integration. Together, our findings establish a translaminar inhibitory recurrence from deep layers that sharpens feature selectivity in superficial cortical layers.

A topographic map of auditory space is a feature found in the superior colliculus (SC) of many species, including CBA/CaJ mice. In this genetic background, high-frequency monaural spectral cues and interaural level differences (ILDs) are used to compute spatial receptive fields (RFs) that form a topographic map along the azimuth. Unfortunately, C57BL/6 mice, a strain widely used for transgenic manipulation, display age-related hearing loss (AHL) because of an inbred mutation in the Cadherin 23 gene ( Cdh23 ) that affects hair cell mechanotransduction. To overcome this problem, researchers have used young C57BL/6 mice in their studies, as they have been shown to have normal hearing thresholds. However, important details of the auditory response characteristics of the SC such as spectral responses and spatial localization, have not been characterized in young C57BL/6 mice. Here, we show that two- to four-month C57BL/6 mice lack neurons with frontal auditory RFs and therefore lack a topographic representation of auditory space in the SC. Analysis of the spectrotemporal RFs (STRFs) of the SC auditory neurons shows that C57BL/6 mouse SC neurons lack the ability to detect the high-frequency (>40 kHz) spectral cues that are needed to compute frontal RFs. We also show that crossing C57BL/6 mice with CBA/CaJ mice or introducing one copy of the wild-type Cdh23 to C57BL/6 mice rescues the high-frequency hearing deficit and improves the topographic map of auditory space. Taken together, these results demonstrate the importance of high-frequency hearing in computing a topographic map of auditory space.