Publications using the ES1 & EC1

The hippocampus has been thought to process auditory information. However, the properties, pathway, and role of hippocampal auditory responses are unclear. With loose-patch recordings, we found that hippocampal neurons are mainly responsive to noise and are not tonotopically organized. Their latencies are shorter than those of primary auditory cortical (A1) neurons but longer than those of medial septal (MS) neurons, suggesting that hippocampal auditory information comes from MS neurons rather than from A1 neurons. Silencing the MS blocks both hippocampal auditory responses and memory of auditory fear conditioning trained with noise and tone. Auditory fear conditioning was associated with some cues but not with a specific frequency of sound, as demonstrated by animals trained with noise, 2.5-, 5-, 10-, 15-, or 30-kHz tones, and tested with these sounds. Therefore, the noise responses of hippocampal neurons have identified a population of neurons that can be associated with auditory fear conditioning.

Cortical neurons are often functionally heterogeneous even for molecularly defined subtypes. In sensory cortices, physiological responses to natural stimuli can be sparse and vary widely even for neighboring neurons. It is thus difficult to parse out circuits that encode specific stimuli for further experimentation. Here, we report the development of a Cre-reporter mouse that allows recombination for cellular labeling and genetic manipulation, and use it with an activity-dependent Fos -CreER T2 driver to identify functionally active circuits in the auditory cortex. In vivo targeted patch recordings validate our method for neurons responding to physiologically relevant natural sounds such as pup wriggling calls and ultrasonic vocalizations (USVs). Using this system to investigate cortical responses in postpartum mothers, we find a transient recruitment of neurons highly responsive to USVs. This subpopulation of neurons has distinct physiological properties that improve the coding efficiency for pup USV calls, implicating it as a unique signature in parental plasticity.

Germline mutations in Mir96 , one of three co-expressed polycistronic miRNA genes ( Mir96, Mir182, Mir183 ), cause hereditary hearing loss in humans and mice. Transgenic FVB/NCrl- Tg(GFAP- Mir183,Mir96,Mir182 )MDW1 mice (Tg 1MDW ), which overexpress this neurosensory-specific miRNA cluster in the inner ear, were developed as a model system to identify, in the aggregate, target genes and biologic processes regulated by the miR-183 cluster. Histological assessments demonstrate Tg 1MDW/1MDW homozygotes have a modest increase in cochlear inner hair cells (IHCs). Affymetrix mRNA microarray data analysis revealed that downregulated genes in P5 Tg 1MDW/1MDW cochlea are statistically enriched for evolutionarily conserved predicted miR-96, miR-182 or miR-183 target sites. ABR and DPOAE tests from 18 days to 3 months of age revealed that Tg 1MDW/1MDW homozygotes develop progressive neurosensory hearing loss that correlates with histologic assessments showing massive losses of both IHCs and outer hair cells (OHCs). This mammalian miRNA misexpression model demonstrates a potency and specificity of cochlear homeostasis for one of the dozens of endogenously co-expressed, evolutionally conserved, small non-protein coding miRNA families. It should be a valuable tool to predict and elucidate miRNA-regulated genes and integrated functional gene expression networks that significantly influence neurosensory cell differentiation, maturation and homeostasis.

In the primary auditory cortex (A1), neuronal ensembles are activated relative to anticipated sound events following rhythmic stimulation, but whether the echo responses of the neurons are related to their frequency selectivity remains unknown. Therefore, we used in vivo two-photon Ca 2+ imaging to record the neuronal activities in the mouse A1 to elucidate the relationship between their echo responses and frequency selectivity. We confirmed the presence of echo responses in a subgroup of mouse Layer 2/3 A1 neurons following a train of rhythmic pure tone stimulation. After testing with a range of frequencies, we found that these echo responses occurred preferentially close to the best frequencies of the neurons. The local organization of the echo responses of the neurons was heterogeneous in the A1. Therefore, these results indicate that the observed echo responses of neurons within A1 are highly related to their frequency selectivity.

The neurodevelopmental disorder Angelman syndrome (AS) is characterized by intellectual disability, motor dysfunction, distinct behavioral aspects, and epilepsy. AS is caused by a loss of the maternally expressed UBE3A gene, and many of the symptoms are recapitulated in a Ube3a mouse model of this syndrome. At the cellular level, changes in the axon initial segment (AIS) have been reported, and changes in vesicle cycling have indicated the presence of presynaptic deficits. Here we studied the role of UBE3A in the auditory system by recording synaptic transmission at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) through in vivo whole cell and juxtacellular recordings. We show that MNTB principal neurons in Ube3a mice exhibit a hyperpolarized resting membrane potential, an increased action potential (AP) amplitude and a decreased AP half width. Moreover, both the pre- and postsynaptic AP in the calyx of Held synapse of Ube3a mice showed significantly faster recovery from spike depression. An increase in AIS length was observed in the principal MNTB neurons of Ube3a mice, providing a possible substrate for these gain-of-function changes. Apart from the effect on APs, we also observed that EPSPs showed decreased short-term synaptic depression (STD) during long sound stimulations in AS mice, and faster recovery from STD following these tones, which is suggestive of a presynaptic gain-of-function. Our findings thus provide in vivo evidence that UBE3A plays a critical role in controlling synaptic transmission and excitability at excitatory synapses.

The auditory function develops and matures after birth in many mammalian species. After hearing onset, environmental sounds exert profound and long-term effects on auditory functions. However, the effects of the acoustic environment on the functional development of the peripheral auditory system, especially the cochlear sensory hair cells, are still unclear. In the present study, we exposed mouse pups to frequency-enriched acoustic environments in postnatal days 0–14. The results indicated that the acoustic environment significantly decreased the threshold of the auditory brainstem response in a frequency-specific manner. Compared with controls, no difference was found in the number and alignment of inner and outer hair cells or in the length of hair bundles after acoustic overstimulation. The expression and function of prestin, the motor protein of outer hair cells (OHCs), were specifically increased in OHCs activated by acoustic stimulation at postnatal days 7–11. We analyzed the postnatal maturation of ribbon synapses in the hair cell areas. After acoustic stimulation, the number of ribbon synapses was closer to the mature stage than to the controls. Taken together, these data indicate that early acoustic exposure could promote the functional maturation of cochlear hair cells and the development of hearing.

We investigated whether visuoauditory association can be artificially implanted in rodents and then retrieved in a behaviorally relevant context. Rats were trained to approach the left or right hole of a behavioral apparatus to retrieve a reward depending on the side of electrical stimulation of the auditory cortex (EAC) they received and mice were fear-conditioned to EAC. Next, an irrelevant visual stimulus (VS) was repeatedly paired with EAC in the presence of cholecystokinin (CCK) or with activation of terminals of entorhinal CCK neurons in the auditory cortex. In subsequent behavioral testing with VS, rats approached the hole associated with reward availability and mice showed a freezing response to the VS. A CCK antagonist blocked the establishment of visuoauditory association, whereas a CCK agonist rescued the deficit of association. Our findings provide a scientific foundation for “memory implantation” and indicate that CCK is the switching chemical for formation of visuoauditory association.

Perception is characterized by a reciprocal exchange of predictions and prediction error signals between neural regions. However, the relationship between such sensory mismatch responses and hierarchical predictive processing has not yet been demonstrated at the neuronal level in the auditory pathway. We recorded single-neuron activity from different auditory centers in anaesthetized rats and awake mice while animals were played a sequence of sounds, designed to separate the responses due to prediction error from those due to adaptation effects. Here we report that prediction error is organized hierarchically along the central auditory pathway. These prediction error signals are detectable in subcortical regions and increase as the signals move towards auditory cortex, which in turn demonstrates a large-scale mismatch potential. Finally, the predictive activity of single auditory neurons underlies automatic deviance detection at subcortical levels of processing. These results demonstrate that prediction error is a fundamental component of singly auditory neuron responses.

The hippocampus is known as a neuronal structure involved in learning, memory and spatial navigation using multi-sensory cues. However, the basic features of its response to acoustic stimuli without any behavioral tasks (conditioning) remains poorly studied. Here, we investigated the CA3 response to auditory stimuli using in vivo loose-patch recordings in awake and anesthetized C57 mice. Different acoustic stimuli in addition to broadband noise such as click, FM sound and pure tone were applied to test the response of CA3 in awake animals. It was found that the wakefulness of the animal is important for the recorded neurons to respond. The CA3 neurons showed a stronger response to broadband noise rather than the other type of stimuli which suggested that auditory information arrived at CA3 via broadband pathways. Finally, we investigated the excitatory and inhibitory inputs to CA3 neurons by using in vivo whole-cell voltage-clamp techniques with the membrane potential holding at -70 and 0 mV, respectively. In awake animals, the excitatory and inhibitory inputs CA3 neurons receive induced by noise are balanced by showing stable intervals and proportional changes of their latencies and peak amplitudes as a function of the stimulation intensities.

Despite decades of microelectrode recordings, fundamental questions remain about how auditory cortex represents sound-source location. Here, we used in vivo 2-photon calcium imaging to measure the sensitivity of layer II/III neurons in mouse primary auditory cortex (A1) to interaural level differences (ILDs), the principal spatial cue in this species. Although most ILD-sensitive neurons preferred ILDs favoring the contralateral ear, neurons with either midline or ipsilateral preferences were also present. An opponent-channel decoder accurately classified ILDs using the difference in responses between populations of neurons that preferred contralateral-ear-greater and ipsilateral-ear-greater stimuli. We also examined the spatial organization of binaural tuning properties across the imaged neurons with unprecedented resolution. Neurons driven exclusively by contralateral ear stimuli or by binaural stimulation occasionally formed local clusters, but their binaural categories and ILD preferences were not spatially organized on a more global scale. In contrast, the sound frequency preferences of most neurons within local cortical regions fell within a restricted frequency range, and a tonotopic gradient was observed across the cortical surface of individual mice. These results indicate that the representation of ILDs in mouse A1 is comparable to that of most other mammalian species, and appears to lack systematic or consistent spatial order.

We generated constitutive knockout mouse models for the α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits by derivation from conditional knockouts by breeding with CRE deleter mice. We then backcrossed them onto a C57BL/6J genetic background. In this manuscript, we report the generation of the strains and an auditory phenotypic characterization of the constitutive α9 and α10 knockouts and a double α9α10 constitutive knockout. Although the α9 and α10 nAChR subunits are relevant to a number of physiological measures, we chose to characterize the mouse with auditory studies to compare them to existing but different α9 and α10 nAChR knockouts (KOs). Auditory brainstem response (ABR) measurements and distortion product otoacoustic emissions (DPOAEs) showed that all constitutive mouse strains had normal hearing. DPOAEs with contralateral noise (efferent adaptation measurements), however, showed that efferent strength was significantly reduced after deletion of both the α9 and α10 subunits, in comparison to wildtype controls. Animals tested were 3-8 weeks of age and efferent strength was not correlated with age. Confocal studies of single and double constitutive KOs showed that all KOs had abnormal efferent innervation of cochlear hair cells. The morphological results are similar to those obtained in other strains using constitutive deletion of exon 4 of α9 or α10 nAChR. The results of our physiological studies, however, differ from previous auditory studies using a α9 KO generated by deletion of the exon 4 region and backcrossed onto a mixed CBA/CaJ X 129Sv background.

The basal forebrain (BF) has long been implicated in attention, learning and memory, and recent studies have established a causal relationship between artificial BF activation and arousal. However, neural ensemble dynamics in the BF still remains unclear. Here, recording neural population activity in the BF and comparing it with simultaneously recorded cortical population under both anesthetized and unanesthetized conditions, we investigate the difference in the structure of spontaneous population activity between the BF and the auditory cortex (AC) in mice. The AC neuronal population show a skewed spike rate distribution, a higher proportion of short (≤80 ms) inter-spike intervals (ISIs) and a rich repertoire of rhythmic firing across frequencies. Although the distribution of spontaneous firing rate in the BF is also skewed, a proportion of short ISIs can be explained by a Poisson model at short time scales (≤20 ms) and spike count correlations are lower compared to AC cells, with optogenetically identified cholinergic cell pairs showing exceptionally higher correlations. Furthermore, a smaller fraction of BF neurons shows spike-field entrainment across frequencies: a subset of BF neurons fire rhythmically at slow (≤6 Hz) frequencies, with varied phase preferences to ongoing field potentials, in contrast to a consistent phase preference of AC populations. Firing of these slow rhythmic BF cells is correlated to a greater degree than other rhythmic BF cell pairs. Overall, the fundamental difference in the structure of population activity between the AC and BF is their temporal coordination, in particular their operational timescales. These results suggest that BF neurons slowly modulate downstream populations whereas cortical circuits transmit signals on multiple timescales. Thus, the characterization of the neural ensemble dynamics in the BF provides further insight into the neural mechanisms, by which brain states are regulated.

Introduction: Permanent hearing loss and tinnitus as side-effects from treatment with the anticancer drug cisplatin is a clinical problem. Ototoxicity may be reduced by co-administration of an otoprotective agent, but the results in humans have so far been modest. Aim: The present preclinical in vivo study aimed to explore the protective efficacy of hydrogen (H2) inhalation on ototoxicity induced by intravenous cisplatin. Materials and Methods: Albino guinea pigs were divided into four groups. The Cispt (n = 11) and Cispt+H2 (n = 11) groups were given intravenous cisplatin (8 mg/kg b.w., injection rate 0.2 ml/min). Immediately after, the Cispt+H2 group also received gaseous H2 (2% in air, 60 min). The H2 group (n = 5) received only H2 and the Control group (n = 7) received neither cisplatin nor H2. Ototoxicity was assessed by measuring frequency specific ABR thresholds before and 96 h after treatment, loss of inner (IHCs) and outer (OHCs) hair cells, and by performing densitometry-based immunohistochemistry analysis of cochlear synaptophysin, organic transporter 2 (OCT2), and copper transporter 1 (CTR1) at 12 and 7 mm from the round window. By utilizing metabolomics analysis of perilymph the change of metabolites in the perilymph was assessed. Results: Cisplatin induced electrophysiological threshold shifts, hair cell loss, and reduced synaptophysin immunoreactivity in the synapse area around the IHCs and OHCs. H2 inhalation mitigated all these effects. Cisplatin also reduced the OCT2 intensity in the inner and outer pillar cells and in the stria vascularis as well as the CTR1 intensity in the synapse area around the IHCs, the Deiters' cells, and the stria vascularis. H2 prevented the majority of these effects. Conclusion: H2 inhalation can reduce cisplatin-induced ototoxicity on functional, cellular, and subcellular levels. It is proposed that synaptopathy may serve as a marker for cisplatin ototoxicity. The effect of H2 on the antineoplastic activity of cisplatin needs to be further explored.

Topographic representation of the receptor surface is a fundamental feature of sensory cortical organization. This is imparted by the thalamus, which relays information from the periphery to the cortex. To better understand the rules governing thalamocortical connectivity and the origin of cortical maps, we used in vivo two-photon calcium imaging to characterize the properties of thalamic axons innervating different layers of mouse auditory cortex. Although tonotopically organized at a global level, we found that the frequency selectivity of individual thalamocortical axons is surprisingly heterogeneous, even in layers 3b/4 of the primary cortical areas, where the thalamic input is dominated by the lemniscal projection. We also show that thalamocortical input to layer 1 includes collaterals from axons innervating layers 3b/4 and is largely in register with the main input targeting those layers. Such locally varied thalamocortical projections may be useful in enabling rapid contextual modulation of cortical frequency representations.

In many excitatory synapses, mobile zinc is found within glutamatergic vesicles and is coreleased with glutamate. Ex vivo studies established that synaptically released (synaptic) zinc inhibits excitatory neurotransmission at lower frequencies of synaptic activity but enhances steady state synaptic responses during higher frequencies of activity. However, it remains unknown how synaptic zinc affects neuronal processing in vivo. Here, we imaged the sound-evoked neuronal activity of the primary auditory cortex in awake mice. We discovered that synaptic zinc enhanced the gain of sound-evoked responses in CaMKII-expressing principal neurons, but it reduced the gain of parvalbumin- and somatostatin-expressing interneurons. This modulation was sound intensity-dependent and, in part, NMDA receptor-independent. By establishing a previously unknown link between synaptic zinc and gain control of auditory cortical processing, our findings advance understanding about cortical synaptic mechanisms and create a new framework for approaching and interpreting the role of the auditory cortex in sound processing.

Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.

Cannabinoid receptors (CBRs) are widely distributed in the brain, including the inferior colliculus (IC). Here, we aim to study whether endocannabinoids influence a specific type of neuronal adaptation, namely, stimulus-specific adaptation (SSA) found in some IC neurons. SSA is important because it has been found as early as the level of the midbrain and therefore it may be a neuronal correlate of early indices of deviance detection. Furthermore, recent studies have demonstrated a direct link between SSA and MMN, that is widely used as an outcome measure in a variety of human neurodegenerative disorders. SSA is considered a form of short-term plasticity, and CBRs have been shown to play a role in short-term neural plasticity. Therefore, it is reasonable to hypothesize that endocannabinoids may play a role in the generation or modulation of SSA. We recorded single units in the IC under an oddball paradigm stimulation. The results demonstrate that cannabinoid agonists lead to a reduction in the neuronal adaptation. This change is due to a differential increase of the neuronal firing rate to the standard tone alone. Furthermore, we show that the effect is mediated by the cannabinoid receptor 1 (CBR1). Thus, cannabinoid agonists down-modulate SSA in IC neurons.

Sound stimuli are characterized by their rich spectral and temporal dynamic properties. Individual neurons in auditory cortex (ACX) encode both spectral and temporal aspects of sounds e.g. sound onset and/or offset. While the different fields of the ACX show gradients of frequency selectivity the large-scale organization of sound dynamics is unknown. We used widefield imaging of GCaMP6s in awake mouse ACX combined with a novel unsupervised image segmentation technique to investigate the spatiotemporal representation of sound onset and offset. Using this technique, we identified known auditory fields but also detected novel ACX areas. Furthermore, we found that ACX areas differed in their responses to tone onset and offset. Multiple areas were preferentially activated by tone offset, and on-response areas were more spatially localized than off-response areas. We also found tonotopy in off-responses. Together our results demonstrate a different spatial distribution of neurons across ACX for processing sound onsets versus offsets.

Hair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.