Electrophysiology publications

Peripheral sensory organ damage leads to compensatory cortical plasticity that is associated with a remarkable recovery of cortical responses to sound. The precise mechanisms that explain how this plasticity is implemented and distributed over a diverse collection of excitatory and inhibitory cortical neurons remain unknown. After noise trauma and persistent peripheral deficits, we found recovered sound-evoked activity in mouse A1 excitatory principal neurons (PNs), parvalbumin- and vasoactive intestinal peptide-expressing neurons (PVs and VIPs), but reduced activity in somatostatin-expressing neurons (SOMs). This cell-type-specific recovery was also associated with cell-type-specific intrinsic plasticity. These findings, along with our computational modelling results, are consistent with the notion that PV plasticity contributes to PN stability, SOM plasticity allows for increased PN and PV activity, and VIP plasticity enables PN and PV recovery by inhibiting SOMs.

The auditory cortex (AC) modulates the activity of upstream pathways in the auditory brainstem via descending (corticofugal) projections. This feedback system plays an important role in the plasticity of the auditory system by shaping response properties of neurons in many subcortical nuclei. The majority of layer (L) 5 corticofugal neurons project to the inferior colliculus (IC). This corticocollicular (CC) pathway is involved in processing of complex sounds, auditory-related learning, and defense behavior. Partly due to their location in deep cortical layers, CC neuron population activity patterns within neuronal AC ensembles remain poorly understood. We employed two-photon imaging to record the activity of hundreds of L5 neurons in anesthetized as well as awake animals. CC neurons are broader tuned than other L5 pyramidal neurons and display weaker topographic order in core AC subfields. Network activity analyses revealed stronger clusters of CC neurons compared to non-CC neurons, which respond more reliable and integrate information over larger distances. However, results obtained from secondary auditory cortex (A2) differed considerably. Here CC neurons displayed similar or higher topography, depending on the subset of neurons analyzed. Furthermore, specifically in A2, CC activity clusters formed in response to complex sounds were spatially more restricted compared to other L5 neurons. Our findings indicate distinct network mechanism of CC neurons in analyzing sound properties with pronounced subfield differences, demonstrating that the topography of sound-evoked responses within AC is neuron-type dependent.

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.

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.

The environment noise may disturb animal behavior and echolocation via three potential mechanisms: acoustic masking, reduced attention and noise avoidance. Compared with the mechanisms of reduced attention and noise avoidance, acoustic masking is thought to occur only when the signal and background noise overlap spectrally and temporally. In this study, we investigated the effects of spectrally non-overlapping noise on echolocation pulses and electrophysiological responses of a constant frequency–frequency modulation (CF-FM) bat, Hipposideros pratti. We found that H. pratti called at higher intensities while keeping the CFs of their echolocation pulses consistent. Electrophysiological tests indicated that the noise could decrease auditory sensitivity and sharp intensity tuning, suggesting that spectrally non-overlapping noise imparts an acoustic masking effect. Because anthropogenic noises are usually concentrated at low frequencies and are spectrally non-overlapping with the bat’s echolocation pulses, our results provide further evidence of negative consequences of anthropogenic noise. On this basis, we sound a warning against noise in the foraging habitats of echolocating bats.

Brain asymmetry in the sensitivity to spectrotemporal modulation is an established functional feature that underlies the perception of speech and music. The left auditory cortex (ACx) is believed to specialize in processing fast temporal components of speech sounds, and the right ACx slower components. However, the circuit features and neural computations behind these lateralized spectrotemporal processes are poorly understood. To answer these mechanistic questions we use mice, an animal model that captures some relevant features of human communication systems. In this study, we screened for circuit features that could subserve temporal integration differences between the left and right ACx. We mapped excitatory input to principal neurons in all cortical layers and found significantly stronger recurrent connections in the superficial layers of the right ACx compared to the left. We hypothesized that the underlying recurrent neural dynamics would exhibit differential characteristic timescales corresponding to their hemispheric specialization. To investigate, we recorded spike trains from awake mice and estimated the network time constants using a statistical method to combine evidence from multiple weak signal-to-noise ratio neurons. We found longer temporal integration windows in the superficial layers of the right ACx compared to the left as predicted by stronger recurrent excitation. Our study shows substantial evidence linking stronger recurrent synaptic connections to longer network timescales. These findings support speech processing theories that purport asymmetry in temporal integration is a crucial feature of lateralization in auditory processing.

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.

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.

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.

Acoustic communication signals are inevitably challenged by ambient noise. In response to noise, many animals adjust their calls to maintain signal detectability. However, the mechanisms by which the auditory system adapts to the adjusted pulses are unclear. Our previous study revealed that the echolocating bat, Hipposideros pratti , increased its pulse intensity in the presence of background white noise. In vivo single-neuron recording demonstrated that the auditory midbrain neurons tuned to the second harmonic (H2 neurons) increased their minimal threshold (MT) to a similar degree as the increment of pulse intensity in the presence of the background noise. Furthermore, the H2 neurons exhibited consistent spike rates at their best amplitudes and sharper intensity tuning with background white noise compared with silent conditions. The previous data indicated that sound intensity analysis by auditory midbrain neurons was adapted to the increased pulse intensity in the same noise condition. This study further examined the echolocation pulse frequency and frequency analysis of auditory midbrain neurons with noise conditions. The data revealed that H. pratti did not shift the resting frequency in the presence of background noise. The auditory midbrain neuronal frequency analysis highly linked to processing the resting frequency with the presence of noise by presenting the constant best frequency (BF), frequency sensitivity, and frequency selectivity. Thus, our results suggested that auditory midbrain neuronal responses in background white noise are adapted to process echolocation pulses in the noise conditions.

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.

Background: Auditory hallucinations (which occur when the distinction between thoughts and perceptions is blurred) are common in psychotic disorders. The orbitofrontal cortex (OFC) may be implicated, because it receives multiple inputs, including sound and affective value via the amygdala, orchestrating complex emotional responses. We aimed to elucidate the circuit and neuromodulatory mechanisms that underlie the processing of emotionally salient auditory stimuli in the OFC — mechanisms that may be involved in auditory hallucinations. Methods: We identified the cortico-cortical connectivity conveying auditory information to the mouse OFC; its sensitivity to neuromodulators involved in psychosis and postpartum depression, such as dopamine and neurosteroids; and its sensitivity to sensory gating (defective in dysexecutive syndromes). Results: Retrograde tracers in OFC revealed input cells in all auditory cortices. Acoustic responses were abolished by pharmacological and chemogenetic inactivation of the above-identified pathway. Acoustic responses in the OFC were reduced by local dopaminergic agonists and neurosteroids. Noticeably, apomorphine action lasted longer in the OFC than in auditory areas, and its effect was modality-specific (augmentation for visual responses), whereas neurosteroid action was sex-specific. Finally, acoustic responses in the OFC reverberated to the auditory association cortex via feedback connections and displayed sensory gating, a phenomenon of local origin, given that it was not detectable in input auditory cortices. Limitations: Although our findings were for mice, connectivity and sensitivity to neuromodulation are conserved across mammals. Conclusion: The corticocortical loop from the auditory association cortex to the OFC is dramatically sensitive to dopamine and neurosteroids. This suggests a clinically testable circuit behind auditory hallucinations. The function of OFC input–output circuits can be studied in mice with targeted and clinically relevant mutations related to their response to emotionally salient sounds.

Brain asymmetry in the sensitivity to spectrotemporal modulation is an established functional feature that underlies the perception of speech and music. Recent studies in humans suggest separable neural mechanisms must underlie these cognitively complex functions. The left Auditory Cortex (ACx) is believed to specialize in processing fast temporal components of speech sounds, and the right ACx slower components. However, the circuit features and neural computations behind these lateralized spectrotemporal processes are poorly understood. To answer these mechanistic questions we use mice, an animal model that captures some relevant features of human communication systems. In this study, we screened for circuit features that could subserve temporal integration differences between the left and right ACx. We mapped excitatory input to principal neurons in all cortical layers and found significantly stronger recurrent connections in the superficial layers of the right ACx compared to the left. We hypothesized that the underlying recurrent neural dynamics would exhibit differential characteristic timescales corresponding to their hemispheric specialization. To investigate, we recorded spike trains from awake mice and estimated the network time constants using a quasi-Bayesian method to combine evidence from multiple weak signal-to-noise ratio neurons. We found longer temporal integration windows in the right ACx compared to the left as predicted by stronger recurrent excitation. Our study shows direct evidence that stronger recurrent synaptic connections lead to longer network time scales. These findings support speech processing theories that purport asymmetric integration time constants is a crucial feature of lateralization in auditory processing.

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.

The orbitofrontal cortex (OFC) controls flexible behavior through stimulus value updating based on stimulus outcome associations, allowing seamless navigation in dynamic sensory environments with changing contingencies. Sensory cue driven responses, primarily studied through behavior, exist in the OFC. However, OFC neurons’ sensory response properties, particularly auditory, are unknown in the mouse, a genetically tractable animal. We show that mouse OFC single neurons have unique auditory response properties showing pure oddball detection and long timescales of adaptation resulting in stimulus-history dependence. Further, we show that OFC auditory responses are shaped by two parallel sources in the auditory thalamus, lemniscal and non-lemniscal. The latter underlies a large component of the observed oddball detection and additionally controls persistent activity in the OFC through the amygdala. The deviant selectivity can serve as a signal for important changes in the auditory environment. Such signals, if coupled with persistent activity, obtained by disinhibitory control from the non-lemniscal auditory thalamus or amygdala, will allow for associations with a delayed outcome related signal, like reward prediction error, and potentially forms the basis of updating stimulus outcome associations in the OFC. Thus, the baseline sensory responses allow the behavioral requirement-based response modification through relevant inputs from other structures related to reward, punishment, or memory. Thus, alterations in these responses in neurologic disorders can lead to behavioral deficits.

Sound localization plays a critical role in animal survival. Three cues can be used to compute sound direction: interaural timing differences (ITDs), interaural level differences (ILDs) and the direction-dependent spectral filtering by the head and pinnae (spectral cues). Little is known about how spectral cues contribute to the neural encoding of auditory space. Here we report on auditory space encoding in the mouse superior colliculus (SC). We show that the mouse SC contains neurons with spatially-restricted receptive fields (RFs) that form an azimuthal topographic map. We found that frontal RFs require spectral cues and lateral RFs require ILDs. The neurons with frontal RFs have frequency tunings that match the spectral structure of the specific head and pinna filter for sound coming from the front. These results demonstrate that patterned spectral cues in combination with ILDs give rise to the topographic map of azimuthal auditory space.

In auditory-conditioned fear learning, the freezing response is independent of the sound frequencies used, but the frequency of the conditioned sound is considered distinct from those of unrelated sounds based on electrophysiological responses in the auditory system. Whether an emergent discriminative learning underlies auditory fear conditioning and which nuclei and pathways are involved in it remain unclear. Using behavioral and electrophysiological assays, we found that the response of medial prefrontal cortex (mPFC) neurons to a conditioned auditory stimulus (CS) was enhanced relative to the response to unrelated frequencies (UFs) after auditory fear conditioning, and mice could distinguish the CS during multifrequency testing, a phenomenon called emergent discriminative learning. After silencing the mPFC with muscimol, emergent discriminative learning was blocked. In addition, the pure tone responses of mPFC neurons were inhibited after injection of lidocaine in the ipsilateral primary auditory cortex (A1), and the emergent discriminative learning was blocked by silencing both sides of A1 with muscimol. This study, therefore, provides evidence for an emergent discriminative learning mediated by mPFC and A1 neurons after auditory fear conditioning.

Although within-modality sensory plasticity is limited to early developmental periods, cross-modal plasticity can occur even in adults. In vivo electrophysiological studies have shown that transient visual deprivation (dark exposure, DE) in adult mice improves the frequency selectivity and discrimination of neurons in thalamorecipient layer 4 (L4) of primary auditory cortex (A1). Since sound information is processed hierarchically in A1 by populations of neurons, we investigated whether DE alters network activity in A1 L4 and layer 2/3 (L2/3). We examined neuronal populations in both L4 and L2/3 using in vivo two-photon calcium (Ca 2+ ) imaging of transgenic mice expressing GCaMP6s. We find that one week of DE in adult mice increased the sound evoked responses and frequency selectivity of both L4 and L2/3 neurons. Moreover, after DE the frequency representation changed with L4 and L2/3 showing a reduced representation of cells with best frequencies (BFs) between 8 and 16 kHz and an increased representation of cells with BFs above 32 kHz. Cells in L4 and L2/3 showed decreased pairwise signal correlations (SCs) consistent with sharper tuning curves. The decreases in SCs were larger in L4 than in L2/3. The decreased pairwise correlations indicate a sparsification of A1 responses to tonal stimuli. Thus, cross-modal experience in adults can both alter the sound-evoked responses of A1 neurons and change activity correlations within A1 potentially enhancing the encoding of auditory stimuli.