Electrophysiology publications

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

Reconstructing axonal projections of single neurons at the whole-brain level is currently a converging goal of the neuroscience community that is fundamental for understanding the logic of information flow in the brain. Thousands of single neurons from different brain regions have recently been morphologically reconstructed, but the corresponding physiological functional features of these reconstructed neurons are unclear. By combining two-photon Ca 2+ imaging with targeted single-cell plasmid electroporation, we reconstruct the brain-wide morphologies of single neurons that are defined by a sound-evoked response map in the auditory cortices (AUDs) of awake mice. Long-range interhemispheric projections can be reliably labelled via co-injection with an adeno-associated virus, which enables enhanced expression of indicator protein in the targeted neurons. Here we show that this method avoids the randomness and ambiguity of conventional methods of neuronal morphological reconstruction, offering an avenue for developing a precise one-to-one map of neuronal projection patterns and physiological functional features.

Sensory hypersensitivity, especially in the auditory system, is a common symptom in Fragile X syndrome (FXS), the most common monogenic form of intellectual disability. However, linking phenotypes across genetic background strains of mouse models has been a challenge and could underly some of the issues with translatability of drug studies to the human condition. This study is the first to characterize the auditory brain stem response (ABR), a minimally invasive physiological readout of early auditory processing that is also used in humans, in a commonly used mouse background strain model of FXS, C57BL/6J. We measured morphological features of pinna and head and used ABR to measure the hearing range, and monaural and binaural auditory responses in hemizygous males, homozygous females, and heterozygous females compared with those in wild-type mice. Consistent with previous study, we showed no difference in morphological parameters across genotypes or sexes. There was no significant difference in hearing range between the sexes or genotypes, however there was a trend towards high frequency hearing loss in male FXS mice. In contrast, female mice with homozygous FXS had a decreased amplitude of wave IV of the monaural ABR, while there was no difference in males for amplitudes and no change in latency of ABR waveforms across sexes and genotypes. Finally, males with FXS had an increased latency of the binaural interaction component (BIC) at 0 interaural timing difference compared with that in wild-type males. These findings further clarify auditory brain stem processing in FXS by adding more information across genetic background strains allowing for a better understanding of shared phenotypes.

Recent studies have established significant anatomical and functional connections between visual areas and primary auditory cortex (A1), which may be important for cognitive processes such as communication and spatial perception. These studies have raised two important questions: First, which cell populations in A1 respond to visual input and/or are influenced by visual context? Second, which aspects of sound encoding are affected by visual context? To address these questions, we recorded single-unit activity across cortical layers in awake mice during exposure to auditory and visual stimuli. Neurons responsive to visual stimuli were most prevalent in the deep cortical layers and included both excitatory and inhibitory cells. The overwhelming majority of these neurons also responded to sound, indicating unimodal visual neurons are rare in A1. Other neurons for which sound-evoked responses were modulated by visual context were similarly excitatory or inhibitory but more evenly distributed across cortical layers. These modulatory influences almost exclusively affected sustained sound-evoked firing rate (FR) responses or spectrotemporal receptive fields (STRFs); transient FR changes at stimulus onset were rarely modified by visual context. Neuron populations with visually modulated STRFs and sustained FR responses were mostly non-overlapping, suggesting spectrotemporal feature selectivity and overall excitability may be differentially sensitive to visual context. The effects of visual modulation were heterogeneous, increasing and decreasing STRF gain in roughly equal proportions of neurons. Our results indicate visual influences are surprisingly common and diversely expressed throughout layers and cell types in A1, affecting nearly one in five neurons overall.

Recent studies have identified sex-differences in auditory physiology and in the susceptibility to noise-induced hearing loss (NIHL). We hypothesize that 17β-estradiol (E2), a known modulator of auditory physiology, may underpin sex-differences in the response to noise trauma. Here, we gonadectomized B6CBAF1/J mice and used a combination of electrophysiological and histological techniques to study the effects of estrogen replacement on peripheral auditory physiology in the absence of noise exposure and on protection from NIHL. Functional analysis of auditory physiology in gonadectomized female mice revealed that E2-treatment modulated the peripheral response to sound in the absence of changes to the endocochlear potential compared to vehicle-treatment. E2-replacement in gonadectomized female mice protected against hearing loss following permanent threshold shift (PTS)- and temporary threshold shift (TTS)-inducing noise exposures. Histological analysis of the cochlear tissue revealed that E2-replacement mitigated outer hair cell loss and cochlear synaptopathy following noise exposure compared to vehicle-treatment. Lastly, using fluorescent in situ hybridization, we demonstrate co-localization of estrogen receptor-2 with type-1C, high threshold spiral ganglion neurons, suggesting that the observed protection from cochlear synaptopathy may occur through E2-mediated preservation of these neurons. Taken together, these data indicate the estrogen signaling pathways may be harnessed for the prevention and treatment of NIHL.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation ( Klhl18 lowf ) and a targeted mutation ( Klhl18 tm1a(KOMP)Wtsi ). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18 lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18 lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

Alzheimer’s disease (AD) is characterized by a progressive loss of memory and cognitive decline. However, the assessment of AD-associated functional and cognitive changes is still a big challenge. Auditory-evoked cortical potential (AECP) is an event-related potential reflecting not only neural activation in the auditory cortex (AC) but also cognitive activity in the brain. In this study, we used the subdermal needle electrodes with the same electrode setting as the auditory brainstem response (ABR) recording and recorded AECP in normal aging CBA/CaJ mice and APP/PS1 AD mice. AECP in mice usually appeared as three positive peaks, i.e., P1, P2, and P3, and three corresponding negative peaks, i.e., N1, N2, and N3. In normal aging CBA mice, the early sensory peaks P1, N1, and P2 were reduced as age increased, whereas the later cognitive peaks N2, P3, and N3 were increased or had no changes with aging. Moreover, the latency of the P1 peak was increased as age increased, although the latencies of later peaks had a significant reduction with aging. In AD mice, peak P1 was significantly reduced in comparison with wild-type (WT) littermates at young ages, proceeding AD phenotype presentation. In particular, the later cognitive peak P3 was diminished after 3 months old, different from the normal aging effect. However, the latencies of AECP peaks in AD mice generally had no significant delay or changes with aging. Finally, consistent with AECP changes, the accumulation of amyloid precursor protein (APP) at the AC was visible in AD mice as early as 2 months old. These data suggest that AECP could serve as an early, non-invasive, and objective biomarker for detecting AD and AD-related dementia (ADRD).

Although auditory harmonic distortion has been demonstrated psychophysically in humans and electrophysiologically in experimental animals, the cellular origin of the mechanical harmonic distortion remains unclear. To demonstrate the outer hair cell-generated harmonics within the organ of Corti, we measured sub-nanometer vibrations of the reticular lamina from the apical ends of the outer hair cells in living gerbil cochleae using a custom-built heterodyne low-coherence interferometer. The harmonics in the reticular lamina vibration are significantly larger and have broader spectra and shorter latencies than those in the basilar membrane vibration. The latency of the second harmonic is significantly greater than that of the fundamental at low stimulus frequencies. These data indicate that the mechanical harmonics are generated by the outer hair cells over a broad cochlear region and propagate from the generation sites to their own best-frequency locations.

Integration of multi-frequency sounds into a unified perceptual object is critical for recognizing syllables in speech. This “feature binding” relies on the precise synchrony of each component’s onset timing, but little is known regarding its neural correlates. We find that multi-frequency sounds prevalent in vocalizations, specifically harmonics, preferentially activate the mouse secondary auditory cortex (A2), whose response deteriorates with shifts in component onset timings. The temporal window for harmonics integration in A2 was broadened by inactivation of somatostatin-expressing interneurons (SOM cells), but not parvalbumin-expressing interneurons (PV cells). Importantly, A2 has functionally connected subnetworks of neurons preferentially encoding harmonic over inharmonic sounds. These subnetworks are stable across days and exist prior to experimental harmonics exposure, suggesting their formation during development. Furthermore, A2 inactivation impairs performance in a discrimination task for coincident harmonics. Together, we propose A2 as a locus for multi-frequency integration, which may form the circuit basis for vocal processing.

Significance Some animals have evolved an adaptive ability for vocalizing and hearing ultrasonic frequencies that are inaudible to humans (>20 kHz). For decades, it has been postulated that animals hear ultrasonic frequencies with cochlear hair cells using an identical set of mechanotransduction molecules in the hair bundles for hearing audible frequencies. Here, we show that mice lacking mechanosensitive PIEZO2 ion channels have difficulty hearing at ultrasonic frequencies but remain remarkably sensitive to audible frequencies. Thus, animals may use a partially different mechanism for sensing ultrasonic sound emissions.

Integration of information across the senses is critical for perception and is a common property of neurons in the cerebral cortex, where it is thought to arise primarily from corticocortical connections. Much less is known about the role of subcortical circuits in shaping the multisensory properties of cortical neurons. We show that stimulation of the whiskers causes widespread suppression of sound-evoked activity in mouse primary auditory cortex (A1). This suppression depends on the primary somatosensory cortex (S1), and is implemented through a descending circuit that links S1, via the auditory midbrain, with thalamic neurons that project to A1. Furthermore, a direct pathway from S1 has a facilitatory effect on auditory responses in higher-order thalamic nuclei that project to other brain areas. Crossmodal corticofugal projections to the auditory midbrain and thalamus therefore play a pivotal role in integrating multisensory signals and in enabling communication between different sensory cortical areas.

Congenital sensorineural hearing loss (SNHL) affects thousands of infants each year and results in significant delays in speech and language development. Previous studies have shown that early exposure to a simple augmented acoustic environment (AAE) can limit the effects of progressive SNHL on hearing sensitivity. However, SNHL is also accompanied by hearing loss that is not assessed on standard audiological examinations, such as reduced temporal processing acuity. To assess whether sound therapy may improve these deficits, a mouse model of congenital SNHL was exposed to simple or temporally complex AAE. The DBA/2J mouse strain develops rapid, base to apex, progressive SNHL beginning at birth and is functionally deaf by six months of age. Hearing sensitivity and auditory brainstem function was measured using otoacoustic emissions, auditory brainstem response (ABR) and extracellular recording from the inferior colliculus (IC) in mice following exposure to 30 d of continuous AAE. Peripheral function and sound sensitivity in auditory midbrain neurons improved following exposure to both types of AAE. However, exposure to a novel, temporally complex AAE more strongly improved a measure of temporal processing acuity, neural gap-in-noise detection in the auditory midbrain. These experiments suggest that targeted sound therapy may be harnessed to improve hearing outcomes for children suffering from congenital SNHL.

The mechanisms that govern thalamocortical transmission are poorly understood. Recent data have shown that sensory stimuli elicit activity in ensembles of cortical neurons that recapitulate stereotyped spontaneous activity patterns. Here, we elucidate a possible mechanism by which gating of patterned population cortical activity occurs. In this study, sensory-evoked all-or-none cortical population responses were observed in the mouse auditory cortex in vivo and similar stochastic cortical responses were observed in a colliculo-thalamocortical brain slice preparation. Cortical responses were associated with decreases in auditory thalamic synaptic inhibition and increases in thalamic synchrony. Silencing of corticothalamic neurons in layer 6 (but not layer 5) or the thalamic reticular nucleus linearized the cortical responses, suggesting that layer 6 corticothalamic feedback via the thalamic reticular nucleus was responsible for gating stochastic cortical population responses. These data implicate a corticothalamic-thalamic reticular nucleus circuit that modifies thalamic neuronal synchronization to recruit populations of cortical neurons for sensory representations.

Neurostimulant drugs or magnetic/electrical stimulation techniques can overcome attention deficits, but these drugs or techniques are weakly beneficial in boosting the learning capabilities of healthy subjects. Here, we report a stimulation technique, mid-infrared modulation (MIM), that delivers mid-infrared light energy through the opened skull or even non-invasively through a thinned intact skull and can activate brain neurons in vivo without introducing any exogeneous gene. Using c-Fos immunohistochemistry, in vivo single-cell electrophysiology and two-photon Ca 2+ imaging in mice, we demonstrate that MIM significantly induces firing activities of neurons in the targeted cortical area. Moreover, mice that receive MIM targeting to the auditory cortex during an auditory associative learning task exhibit a faster learning speed (~50% faster) than control mice. Together, this non-invasive, opsin-free MIM technique is demonstrated with potential for modulating neuronal activity.

Survival in a dynamic environment requires animals to plan future actions based on past sensory evidence, known as motor planning. However, the neuronal circuits underlying this crucial brain function remain elusive. Here, we employ projection-specific imaging and perturbation methods to investigate the direct pathway linking two key nodes in the motor planning network, the secondary motor cortex (M2) and the midbrain superior colliculus (SC), in mice performing a memory-dependent perceptual decision task. We find dynamic coding of choice information in SC-projecting M2 neurons during motor planning and execution, and disruption of this information by inhibiting M2 terminals in SC selectively impaired decision maintenance. Furthermore, we show that while both excitatory and inhibitory SC neurons receive synaptic inputs from M2, these SC subpopulations display differential temporal patterns in choice coding during behavior. Our results reveal the dynamic recruitment of the premotor-collicular pathway as a circuit mechanism for motor planning.

Detecting the direction of frequency modulation (FM) is essential for vocal communication in both animals and humans. Direction-selective firing of neurons in the primary auditory cortex (A1) has been classically attributed to temporal offsets between feedforward excitatory and inhibitory inputs. However, it remains unclear how cortical recurrent circuitry contributes to this computation. Here, we used two-photon calcium imaging and whole-cell recordings in awake mice to demonstrate that direction selectivity is not caused by temporal offsets between synaptic currents, but by an asymmetry in total synaptic charge between preferred and non-preferred directions. Inactivation of cortical somatostatin-expressing interneurons (SOM cells) reduced direction selectivity, revealing its cortical contribution. Our theoretical models showed that charge asymmetry arises due to broad spatial topography of SOM cell-mediated inhibition which regulates signal amplification in strongly recurrent circuitry. Together, our findings reveal a major contribution of recurrent network dynamics in shaping cortical tuning to behaviorally relevant complex sounds.

Polychlorinated biphenyls (PCBs) are enduring environmental toxicants and exposure is associated with neurodevelopmental deficits. The auditory system appears particularly sensitive, as previous work has shown that developmental PCB exposure causes both hearing loss and gross disruptions in the organization of the rat auditory cortex. However, the mechanisms underlying PCB-induced changes are not known, nor is it known whether the central effects of PCBs are a consequence of peripheral hearing loss. Here, we study changes in both peripheral and central auditory function in rats with developmental PCB exposure using a combination of optical and electrophysiological approaches. Female rats were exposed to an environmental PCB mixture in utero and until weaning. At adulthood, auditory brainstem responses (ABRs) were measured, and synaptic currents were recorded in slices from auditory cortex layer 2/3 neurons. Spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) were more frequent in PCB-exposed rats compared with controls and the normal relationship between IPSC parameters and peripheral hearing was eliminated in PCB-exposed rats. No changes in spontaneous EPSCs were found. Conversely, when synaptic currents were evoked by laser photostimulation of caged-glutamate, PCB exposure did not affect evoked inhibitory transmission, but increased the total excitatory charge, the number and distance of sites that evoke a significant response. Together, these findings indicate that early developmental exposure to PCBs causes long-lasting changes in both inhibitory and excitatory neurotransmission in the auditory cortex that are independent of peripheral hearing changes, suggesting the effects are because of the direct impact of PCBs on the developing auditory cortex.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.

Anxiety disorders involve distorted perception of the world including increased saliency of stress-associated cues. However, plasticity in the initial sensory regions of the brain following a fearful experience has never been examined. The cochlear nucleus (CN) is the first station in the central auditory system, with heterogeneous collections of neurons that not only project to but also receive projections from cortico-limbic regions, suggesting a potential for experience-dependent plasticity. Using wireless neural recordings in freely behaving rats, we demonstrate for the first time that neural gain in the CN is significantly altered by fear conditioning to auditory sequences. Specifically, the ventral subnuclei significantly increased firing rate to the conditioned tone sequence, while the dorsal subnuclei significantly decreased firing rate during the conditioning session overall. These findings suggest subregion-specific changes in the balance of inhibition and excitation in the CN as a result of conditioning experience. Heart rate was measured as the conditioned response (CR), which showed that while pre-conditioned stimulus (CS) responding did not change across baseline and conditioning sessions, significant changes in heart rate were observed to the tone sequence followed by shock. Heart-rate findings support acquisition of conditioned fear. Taken together, the present study presents first evidence for potential experience-dependent changes in auditory perception that involve novel plasticity within the first site of processing auditory information in the brain.

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca 2+ imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.