Altered population activity and local tuning heterogeneity in auditory cortex of Cacna2d3-deficient mice.

The α2δ3 auxiliary subunit of voltage-activated calcium channels is required for normal synaptic transmission and precise temporal processing of sounds in the auditory brainstem. In mice its loss additionally leads to an inability to distinguish amplitude-modulated tones. Furthermore, loss of function of α2δ3 has been associated with autism spectrum disorder in humans. To investigate possible alterations of network activity in the higher-order auditory system in α2δ3 knockout mice, we analyzed neuronal activity patterns and topography of frequency tuning within networks of the auditory cortex (AC) using two-photon Ca2+ imaging. Compared to wild-type mice we found distinct subfield-specific alterations in the primary auditory cortex, expressed in overall lower correlations between the network activity patterns in response to different sounds as well as lower reliability of these patterns upon repetitions of the same sound. Higher AC subfields did not display these alterations but showed a higher amount of well-tuned neurons along with lower local heterogeneity of the neurons' frequency tuning. Our results provide new insight into AC network activity alterations in an autism spectrum disorder-associated mouse model.

α2δ3 is essential for normal structure and function of auditory nerve synapses and is a novel candidate for auditory processing disorders.

The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3(-/-) mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca(2+) channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3(-/-) mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.

Quantitative analysis of neuronal response properties in primary and higher-order auditory cortical fields of awake house mice (Mus musculus).

Because of its great genetic potential, the mouse (Mus musculus) has become a popular model species for studies on hearing and sound processing along the auditory pathways. Here, we present the first comparative study on the representation of neuronal response parameters to tones in primary and higher-order auditory cortical fields of awake mice. We quantified 12 neuronal properties of tone processing in order to estimate similarities and differences of function between the fields, and to discuss how far auditory cortex (AC) function in the mouse is comparable to that in awake monkeys and cats. Extracellular recordings were made from 1400 small clusters of neurons from cortical layers III/IV in the primary fields AI (primary auditory field) and AAF (anterior auditory field), and the higher-order fields AII (second auditory field) and DP (dorsoposterior field). Field specificity was shown with regard to spontaneous activity, correlation between spontaneous and evoked activity, tone response latency, sharpness of frequency tuning, temporal response patterns (occurrence of phasic responses, phasic-tonic responses, tonic responses, and off-responses), and degree of variation between the characteristic frequency (CF) and the best frequency (BF) (CF-BF relationship). Field similarities were noted as significant correlations between CFs and BFs, V-shaped frequency tuning curves, similar minimum response thresholds and non-monotonic rate-level functions in approximately two-thirds of the neurons. Comparative and quantitative analyses showed that the measured response characteristics were, to various degrees, susceptible to influences of anesthetics. Therefore, studies of neuronal responses in the awake AC are important in order to establish adequate relationships between neuronal data and auditory perception and acoustic response behavior.

Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing.

Large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels in inner hair cells (IHCs) of the cochlea are essential for hearing. However, germline deletion of BKα, the pore-forming subunit KCNMA1 of the BK channel, surprisingly did not affect hearing thresholds in the first postnatal weeks, even though altered IHC membrane time constants, decreased IHC receptor potential alternating current/direct current ratio, and impaired spike timing of auditory fibers were reported in these mice. To investigate the role of IHC BK channels for central auditory processing, we generated a conditional mouse model with hair cell-specific deletion of BKα from postnatal day 10 onward. This had an unexpected effect on temporal coding in the central auditory system: neuronal single and multiunit responses in the inferior colliculus showed higher excitability and greater precision of temporal coding that may be linked to the improved discrimination of temporally modulated sounds observed in behavioral training. The higher precision of temporal coding, however, was restricted to slower modulations of sound and reduced stimulus-driven activity. This suggests a diminished dynamic range of stimulus coding that is expected to impair signal detection in noise. Thus, BK channels in IHCs are crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment.

Auditory discrimination learning and knowledge transfer in mice depends on task difficulty.

Mice reproduce interesting effects in auditory discrimination learning and knowledge transfer discussed in human studies: (i) the advantage in the transfer from a hard to an easy task by benefits from transfer of procedural knowledge and information-integration learning, and (ii) the disadvantage in the transfer from easy to hard tasks by inability to generalize across perceptually different classes of stimuli together with initially unsuccessful attempts to transfer cognitive skills from one task to the other. House mice (NMRI strain) were trained in a shuttle-box stimulus discrimination task. They had to discriminate either between two pure tones of different frequencies (PT) or between two different modulation frequencies of an amplitude-modulated tone (AM). Then transfer of knowledge between these two tasks was tested. Mice rapidly learned PT discrimination within two to three training sessions (easy task). AM discrimination learning took longer and did not reach the high performance level of PT discrimination (hard task). No knowledge transfer was detected in animals first trained with the easy (PT) followed by the hard (AM) discrimination task. Mice benefited, however, from knowledge transfer when the AM discrimination was followed by the PT discrimination. When the task changed, confusion of conditioned stimuli occurred if the carrier frequency of the AM was the same as one of the frequencies in the PT task. These results show a hard-to-easy effect when possible knowledge transfer is tested between qualitatively different stimulus classes. The data establish mice as promising animal model for research on genetics of auditory perception and learning.

Impaired Subcortical Processing of Amplitude-Modulated Tones in Mice Deficient for Cacna2d3, a Risk Gene for Autism Spectrum Disorders in Humans.

Temporal processing of complex sounds is a fundamental and complex task in hearing and a prerequisite for processing and understanding vocalization, speech, and prosody. Here, we studied response properties of neurons in the inferior colliculus (IC) in mice lacking Cacna2d3, a risk gene for autism spectrum disorders (ASDs). The α2δ3 auxiliary Ca2+ channel subunit encoded by Cacna2d3 is essential for proper function of glutamatergic synapses in the auditory brainstem. Recent evidence has shown that much of auditory feature extraction is performed in the auditory brainstem and IC, including processing of amplitude modulation (AM). We determined both spectral and temporal properties of single- and multi-unit responses in the IC of anesthetized mice. IC units of α2δ3-/- mice showed normal tuning properties yet increased spontaneous rates compared with α2δ3+/+ When stimulated with AM tones, α2δ3-/- units exhibited less precise temporal coding and reduced evoked rates to higher modulation frequencies (fm). Whereas first spike latencies (FSLs) were increased for only few modulation frequencies, population peak latencies were increased for fm ranging from 20 to 100 Hz in α2δ3-/- IC units. The loss of precision of temporal coding with increasing fm from 70 to 160 Hz was characterized using a normalized offset-corrected (Pearson-like) correlation coefficient, which appeared more appropriate than the metrics of vector strength. The processing deficits of AM sounds analyzed at the level of the IC indicate that α2δ3-/- mice exhibit a subcortical auditory processing disorder (APD). Similar deficits may be present in other mouse models for ASDs.

Long-range effects of GABAergic inhibition in gerbil primary auditory cortex.

Throughout the literature, the effects of iontophoretically applied neurotransmitter agonists or antagonists on the local activity of neurons are typically studied at the site of drug application. Recently, we have demonstrated long-range inhibitory interactions within the primary auditory cortex (AI) that are effective in complex acoustic situations. To further characterize this long-range functional connectivity, we here report the effects of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the GABA(A) antagonist gabazine (SR 95531) on neuronal activity as a function of distance from the application site reaching beyond the diffusion radius of the applied drug. Neuronal responses to pure tone stimulation were simultaneously recorded at the application site and four additional sites, at distances between 300 and 1350 microm from the application site. We found that whereas application of GABA during best frequency (BF) stimulation in general led to a decrease, and gabazine to an increase, in neuronal activity at the application site, a considerable number of units at remote recording sites showed effects opposite to these local, drug-induced effects. These effects were seen both in spiking activity and in amplitudes of local field potentials. At all locations, the effects varied as a function of pure tone stimulation frequency, pointing to a Mexican-hat-like input function resulting from thalamic inputs to the BF region of the cortical neurons and intracortical interconnections projecting to off-BF regions of the neurons. These data demonstrate the existence of long-range, inhibitory interactions within the gerbil AI, realized either by long-range inhibitory projections or by long-range excitatory projections to local inhibitory interneurons.

Differential effects of iontophoretic application of the GABAA-antagonists bicuculline and gabazine on tone-evoked local field potentials in primary auditory cortex: interaction with ketamine anesthesia.

gamma-Aminobutric acid (GABA) is one of the main inhibitory transmitters in the central nervous system. In a recent study we have demonstrated differential effects of two iontophoretically applied GABA(A)-blockers, bicuculline (BIC) and gabazine (SR 95531), on neuronal responses in primary auditory cortex (AI): Whereas the only effect of gabazine was to block GABA(A)-mediated inhibition, BIC application additionally induced dose-dependent side effects, probably on calcium-dependent potassium channels. Here we investigated the effects of the two drugs on pure tone-evoked local field potentials (LFPs) in AI. In contrast to spiking activity, which reflects neuronal output, LFP are believed to mainly reflect dendritic activity and therefore neuronal input. LFPs were recorded from the left AI of anaesthetized and unanaesthetized Mongolian gerbils before, during and after microiontophoretic application of BIC and gabazine using multi-barrel glass electrodes. After the application of both drugs, a significant increase of the amplitude of the N1 component of the LFP was observed in both anaesthetized and unanaesthetized animals, but this increase was significantly more pronounced after BIC than after gabazine application, a result which corresponds to the effects on neuronal discharge rate reported earlier. In contrast, the effects of BIC and gabazine on LFP duration (prolongation) and LFP spectral tuning (sharpening) were affected by ketamine anesthesia, an effect that was not seen in the spiking data. We conclude from the data presented that the main functional role of GABA(A)-mediated inhibition in auditory cortex is to (1) prevent over-excitation (seizures) of cortical networks and (2) to speed up cortical processing.

Electrical stimulation of the midbrain excites the auditory cortex asymmetrically.

BACKGROUND: Auditory midbrain implant users cannot achieve open speech perception and have limited frequency resolution. It remains unclear whether the spread of excitation contributes to this issue and how much it can be compensated by current-focusing, which is an effective approach in cochlear implants. OBJECTIVE: The present study examined the spread of excitation in the cortex elicited by electric midbrain stimulation. We further tested whether current-focusing via bipolar and tripolar stimulation is effective with electric midbrain stimulation and whether these modes hold any advantage over monopolar stimulation also in conditions when the stimulation electrodes are in direct contact with the target tissue. METHODS: Using penetrating multielectrode arrays, we recorded cortical population responses to single pulse electric midbrain stimulation in 10 ketamine/xylazine anesthetized mice. We compared monopolar, bipolar, and tripolar stimulation configurations with regard to the spread of excitation and the characteristic frequency difference between the stimulation/recording electrodes. RESULTS: The cortical responses were distributed asymmetrically around the characteristic frequency of the stimulated midbrain region with a strong activation in regions tuned up to one octave higher. We found no significant differences between monopolar, bipolar, and tripolar stimulation in threshold, evoked firing rate, or dynamic range. CONCLUSION: The cortical responses to electric midbrain stimulation are biased towards higher tonotopic frequencies. Current-focusing is not effective in direct contact electrical stimulation. Electrode maps should account for the asymmetrical spread of excitation when fitting auditory midbrain implants by shifting the frequency-bands downward and stimulating as dorsally as possible.

Visualization of the auditory pathway in rats with 18F-FDG PET activation studies based on different auditory stimuli and reference conditions including cochlea ablation.

Activation studies with positron emission tomography (PET) in auditory implant users explained some of the mechanisms underlying the variability of achieved speech comprehension. Since future developments of auditory implants will include studies in rodents, we aimed to inversely translate functional PET imaging to rats. In normal hearing rats, activity in auditory and non-auditory regions was studied using 18F-fluorodeoxyglucose (18F-FDG) PET with 3 different acoustic conditions: sound attenuated laboratory background, continuous white noise and rippled noise. Additionally, bilateral cochlea ablated animals were scanned. 3D image data were transferred into a stereotaxic standard space and evaluated using volume of interest (VOI) analyses and statistical parametric mapping (SPM). In normal hearing rats alongside the auditory pathway consistent activations of the nucleus cochlearis (NC), olivary complex (OC) and inferior colliculus (IC) were seen comparing stimuli with background. In this respect, no increased activation could be detected in the auditory cortex (AC), which even showed deactivation with white noise stimulation. Nevertheless, higher activity in the AC in normal hearing rats was observed for all 3 auditory conditions against the cochlea ablated status. Vice versa, in ablated status activity in the olfactory nucleus (ON) was higher compared to all auditory conditions in normal hearing rats. Our results indicate that activations can be demonstrated in normal hearing animals based on 18F-FDG PET in nuclei along the central auditory pathway with different types of noise stimuli. However, in the AC missing activation with respect to the background advises the need for more rigorous background noise attenuation for non-invasive reference conditions. Finally, our data suggest cross-modal activation of the olfactory system following cochlea ablation-underlining, that 18F-FDG PET appears to be well suited to study plasticity in rat models for cochlear implantation.

Cortical and subcortical sides of auditory rhythms and pitches.

It is commonly assumed that different perceptual qualities arising from sensory stimuli depend on their physical nature being transformed by specific peripheral receptors, for example, colour, vibration or heat. A notable unexplained exception is the low and high repetition rates of any sound perceived as rhythm or pitch, respectively. Using auditory discrimination learning in bilaterally auditory cortex ablated animals, we demonstrate that the perceptual quality of sounds depends on the way the brain processes stimuli rather than on their physical nature. In this context, cortical and subcortical processing steps have different roles in analysing different aspects of sounds with the complete analysis accomplished not before information converges in the auditory cortex.

Differential effects of iontophoretic in vivo application of the GABA(A)-antagonists bicuculline and gabazine in sensory cortex.

We have compared the effects of microiontophoretic application of the GABA(A)-receptor antagonists bicuculline (BIC) and gabazine (SR95531) on responses to pure tones and to sinusoidally amplitude-modulated (AM) tones in cells recorded extracellularly from primary auditory cortex (AI) of Mongolian gerbils. Besides similar effects in increasing spontaneous and stimulus-evoked activity and their duration, both drugs elicited differential effects on spectral tuning and synchronized responses to AM tones. In contrast to gabazine, iontophoresis of the less potent GABA(A)-antagonist BIC often resulted in substantial broadening of frequency tuning for pure tones and an elimination of synchronized responses to AM tones, particularly with high ejecting currents. BIC-induced effects which could not be replicated by application of gabazine were presumably due to the well-documented, non-GABAergic side-effects of BIC on calcium-dependent potassium channels. Our results thus provide strong evidence that GABA(A)-mediated inhibition in AI does not sharpen frequency tuning for pure tones, but rather contributes to the processing of fast temporal modulations of sound envelopes. They also demonstrate that BIC can have effects on neuronal response selectivity which are not due to blockade of GABAergic inhibition. The results have profound implications for microiontophoretic studies of the role of intracortical inhibition in sensory cortex.

Foxp2 mutations impair auditory-motor association learning.

Heterozygous mutations of the human FOXP2 transcription factor gene cause the best-described examples of monogenic speech and language disorders. Acquisition of proficient spoken language involves auditory-guided vocal learning, a specialized form of sensory-motor association learning. The impact of etiological Foxp2 mutations on learning of auditory-motor associations in mammals has not been determined yet. Here, we directly assess this type of learning using a newly developed conditioned avoidance paradigm in a shuttle-box for mice. We show striking deficits in mice heterozygous for either of two different Foxp2 mutations previously implicated in human speech disorders. Both mutations cause delays in acquiring new motor skills. The magnitude of impairments in association learning, however, depends on the nature of the mutation. Mice with a missense mutation in the DNA-binding domain are able to learn, but at a much slower rate than wild type animals, while mice carrying an early nonsense mutation learn very little. These results are consistent with expression of Foxp2 in distributed circuits of the cortex, striatum and cerebellum that are known to play key roles in acquisition of motor skills and sensory-motor association learning, and suggest differing in vivo effects for distinct variants of the Foxp2 protein. Given the importance of such networks for the acquisition of human spoken language, and the fact that similar mutations in human FOXP2 cause problems with speech development, this work opens up a new perspective on the use of mouse models for understanding pathways underlying speech and language disorders.

AP-2δ is a crucial transcriptional regulator of the posterior midbrain.

Ap-2 transcription factors comprise a family of 5 closely related sequence-specific DNA binding proteins that play pivotal and non-redundant roles in embryonic organogenesis. To investigate the function of Ap-2δ, wδe analyzed its expression during embryogenesis and generated Ap-2δ-deficient mice. In line with the specific expression pattern of Ap-2δ in the mesencephalic tectum and the dorsal midbrain, Ap-2δ-deficient mice failed to maintain the colliculus inferior, a derivative of the dorsal midbrain, as a consequence of increased apoptotic cell death. To identify specific Ap-2δ target genes in cells of the developing dorsal midbrain, we performed whole genome analysis of cDNA expression levels. This approach identified a set of 12 putative target genes being expressed in the developing midbrain, including the transcription factors Pitx2, Mef2c, Bhlhb4 and Pou4f3. Using chromatin immunoprecipitation (CHIP) we showed that some of these genes are direct targets of Ap-2δ. Consistently, we demonstrate that Ap-2δ occupies and activates the Pou4f3 and Bhlhb4 promoters. In addition, known Pou4f3 target genes were downregulated in the posterior midbrain of Ap-2δ-deficient mice. Despite the absence of a central part of the auditory pathway, the presence of neuronal responses to sounds in the neocortex of Ap-2δ-deficient mice indicates that auditory information from the brainstem still reaches the neocortex. In summary, our data define Ap-2δ as an important transcription factor, specifying gene expression patterns required for the development of the posterior midbrain.

Improvement of auditory discrimination learning by Ginkgo biloba extract EGb 761.

The effect of oral application of Ginkgo biloba extract EGb 761 on auditory discrimination learning in Mongolian gerbils was investigated using discrimination tasks with three different degrees of difficulty and two protocols for administration starting 2 weeks prior to or at the beginning of training. In comparison to placebo-treated controls we observed significant improvement of learning performance in EGb 761 treated gerbils in discrimination tasks of all degrees of difficulty, from the easiest to the most demanding. EGb 761 has been reported to increase the extracellular concentration of dopamine in prefrontal cortex of rats which plays a major role in the type of discrimination learning used in the present study. We, therefore, suppose that EGb 761 improves discrimination learning through its effect on the dopaminergic system.

Modified sound-evoked brainstem potentials in Foxp2 mutant mice.

Heterozygous mutations of the human FOXP2 gene cause a developmental disorder involving impaired learning and production of fluent spoken language. Previous investigations of its aetiology have focused on disturbed function of neural circuits involved in motor control. However, Foxp2 expression has been found in the cochlea and auditory brain centers and deficits in auditory processing could contribute to difficulties in speech learning and production. Here, we recorded auditory brainstem responses (ABR) to assess two heterozygous mouse models carrying distinct Foxp2 point mutations matching those found in humans with FOXP2-related speech/language impairment. Mice which carry a Foxp2-S321X nonsense mutation, yielding reduced dosage of Foxp2 protein, did not show systematic ABR differences from wildtype littermates. Given that speech/language disorders are observed in heterozygous humans with similar nonsense mutations (FOXP2-R328X), our findings suggest that auditory processing deficits up to the midbrain level are not causative for FOXP2-related language impairments. Interestingly, however, mice harboring a Foxp2-R552H missense mutation displayed systematic alterations in ABR waves with longer latencies (significant for waves I, III, IV) and smaller amplitudes (significant for waves I, IV) suggesting that either the synchrony of synaptic transmission in the cochlea and in auditory brainstem centers is affected, or fewer auditory nerve fibers and fewer neurons in auditory brainstem centers are activated compared to wildtypes. Therefore, the R552H mutation uncovers possible roles for Foxp2 in the development and/or function of the auditory system. Since ABR audiometry is easily accessible in humans, our data call for systematic testing of auditory functions in humans with FOXP2 mutations.

Auditory cortical contrast enhancing by global winner-take-all inhibitory interactions.

Brains decompose the world into discrete objects of perception, thereby facing the problem of how to segregate and selectively address similar objects that are concurrently present in a scene. Theoretical models propose that this could be achieved by neuronal implementations of so-called winner-take-all algorithms where neuronal representations of objects or object features interact in a competitive manner. Here we present evidence for the existence of such a mechanism in an animal species. We present electrophysiological, neuropharmacological and neuroanatomical data which suggest a novel view of the role of GABA(A)-mediated inhibition in primary auditory cortex (AI), where intracortical GABA(A)-mediated inhibition operates on a global scale within a circular map of sound periodicity representation in AI, with functionally inhibitory projections of similar effect from any location throughout the whole map. These interactions could underlie the proposed competitive "winner-take-all" algorithm to support object segregation, e.g., segregation of different speakers in cocktail-party situations.