Electrophysiology publications

Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.

Despite the absence of tympanic middle ears, snakes can hear. They are thought to primarily detect substrate vibration via connections between the lower jaw and the inner ear. We used the western rat snake ( Pantherophis obsoletus ) to determine how vibration is processed in the brain. We measured vibration‐evoked potential recordings to reveal sensitivity to low‐frequency vibrations. We then used tract tracing combined with immunohistochemistry and Nissl staining to describe the central projections of the papillar branch of the VIIIth nerve. Applications of biotinylated dextran amine to the basilar papilla (homologous to the organ of Corti of mammals) labeled bouton‐like terminals in two first‐order cochlear nuclei, a rostrolateral nucleus angularis (NA) and a caudomedial nucleus magnocellularis (NM). NA formed a distinct dorsal eminence, consisted of heterogenous cell types, and was parvalbumin positive. NM was smaller and poorly separated from the surrounding vestibular nuclei. NM was distinguished by positive calbindin label and included fusiform and round cells. Thus, the atympanate western rat snake shares similar first‐order projections to tympanate reptiles. Auditory pathways may be used for detecting vibration, not only in snakes but also potentially in atympanate early tetrapods.

The perception of pitch is a fundamental percept, which is mediated by the auditory system, requiring the abstraction of stimulus properties related to the spectro-temporal structure of sound. Despite its importance, there is still debate as to the precise areas responsible for its encoding, which may be due to species differences or differences in the recording measures and choices of stimuli used in previous studies. Moreover, it was unknown whether the human brain contains pitch neurons and how distributed such neurons might be. Here, we present the first study to measure multiunit neural activity in response to pitch stimuli in the auditory cortex of intracranially implanted humans. The stimulus sets were regular-interval noise with a pitch strength that is related to the temporal regularity and a pitch value determined by the repetition rate and harmonic complexes. Specifically, we demonstrate reliable responses to these different pitch-inducing paradigms that are distributed throughout Heschl’s gyrus, rather than being localized to a particular region, and this finding was evident regardless of the stimulus presented. These data provide a bridge across animal and human studies and aid our understanding of the processing of a critical percept associated with acoustic stimuli.

This protocol was used to collect data now published in the Journal of Neural Engineering, Spatially selective stimulation of the pig vagus nerve to modulate target effect versus side effect using the multi-contact ImThera stimulating device. https://doi.org/10.1088/1741-2552/acb3fd Dataset has been published to pennsieve.io and can be found at: https://doi.org/10.26275/efbj-8evl

Parallel multisite recordings in the visual cortex of trained monkeys revealed that the responses of spatially distributed neurons to natural scenes are ordered in sequences. The rank order of these sequences is stimulus-specific and maintained even if the absolute timing of the responses is modified by manipulating stimulus parameters. The stimulus specificity of these sequences was highest when they were evoked by natural stimuli and deteriorated for stimulus versions in which certain statistical regularities were removed. This suggests that the response sequences result from a matching operation between sensory evidence and priors stored in the cortical network. Decoders trained on sequence order performed as well as decoders trained on rate vectors but the former could decode stimulus identity from considerably shorter response intervals than the latter. A simulated recurrent network reproduced similarly structured stimulus-specific response sequences, particularly once it was familiarized with the stimuli through non-supervised Hebbian learning. We propose that recurrent processing transforms signals from stationary visual scenes into sequential responses whose rank order is the result of a Bayesian matching operation. If this temporal code were used by the visual system it would allow for ultrafast processing of visual scenes.

Previous work has demonstrated that performance in an auditory selective attention task can be enhanced or impaired, depending on whether a task-irrelevant visual stimulus is temporally coherent with a target auditory stream or with a competing distractor. However, it remains unclear how audiovisual (AV) temporal coherence and auditory selective attention interact at the neurophysiological level. Here, we measured neural activity using EEG while human participants (men and women) performed an auditory selective attention task, detecting deviants in a target audio stream. The amplitude envelope of the two competing auditory streams changed independently, while the radius of a visual disk was manipulated to control the AV coherence. Analysis of the neural responses to the sound envelope demonstrated that auditory responses were enhanced largely independently of the attentional condition: both target and masker stream responses were enhanced when temporally coherent with the visual stimulus. In contrast, attention enhanced the event-related response evoked by the transient deviants, largely independently of AV coherence. These results provide evidence for dissociable neural signatures of bottom-up (coherence) and top-down (attention) effects in AV object formation. SIGNIFICANCE STATEMENT Temporal coherence between auditory stimuli and task-irrelevant visual stimuli can enhance behavioral performance in auditory selective attention tasks. However, how audiovisual temporal coherence and attention interact at the neural level has not been established. Here, we measured EEG during a behavioral task designed to independently manipulate audiovisual coherence and auditory selective attention. While some auditory features (sound envelope) could be coherent with visual stimuli, other features (timbre) were independent of visual stimuli. We find that audiovisual integration can be observed independently of attention for sound envelopes temporally coherent with visual stimuli, while the neural responses to unexpected timbre changes are most strongly modulated by attention. Our results provide evidence for dissociable neural mechanisms of bottom-up (coherence) and top-down (attention) effects on audiovisual object formation.

Our continuous visual experience in daily life is dominated by change. Previous research has focused on visual change due to stimulus motion, eye movements or unfolding events, but not their combined impact across the brain, or their interactions with semantic novelty. We investigate the neural responses to these sources of novelty during film viewing. We analyzed intracranial recordings in humans across 6328 electrodes from 23 individuals. Responses associated with saccades and film cuts were dominant across the entire brain. Film cuts at semantic event boundaries were particularly effective in the temporal and medial temporal lobe. Saccades to visual targets with high visual novelty were also associated with strong neural responses. Specific locations in higher-order association areas showed selectivity to either high or low-novelty saccades. We conclude that neural activity associated with film cuts and eye movements is widespread across the brain and is modulated by semantic novelty.

The subthalamic nucleus (STN) is hypothesized to play a central role in neural processes that regulate self-control. Still uncertain, however, is how that brain structure participates in the dynamically evolving estimation of value that underlies the ability to delay gratification and wait patiently for a gain. To address that gap in knowledge, we studied the spiking activity of neurons in the STN of monkeys during a task in which animals were required to remain motionless for varying periods of time in order to obtain food reward. At the single-neuron and population levels, we found a cost–benefit integration between the desirability of the expected reward and the imposed delay to reward delivery, with STN signals that dynamically combined both attributes of the reward to form a single integrated estimate of value. This neural encoding of subjective value evolved dynamically across the waiting period that intervened after instruction cue. Moreover, this encoding was distributed inhomogeneously along the antero-posterior axis of the STN such that the most dorso-posterior-placed neurons represented the temporal discounted value most strongly. These findings highlight the selective involvement of the dorso-posterior STN in the representation of temporally discounted rewards. The combination of rewards and time delays into an integrated representation is essential for self-control, the promotion of goal pursuit, and the willingness to bear the costs of time delays.

Stressful events can have lasting and impactful effects on behavior, especially by disrupting normal regulation of fear and reward processing. Accurate discrimination among environmental cues predicting threat, safety or reward adaptively guides behavior. Post-traumatic stress disorder (PTSD) represents a condition in which maladaptive fear persists in response to explicit safety-predictive cues that coincide with previously learned threat cues, but without threat being present. Since both the infralimbic cortex (IL) and amygdala have each been shown to be important for fear regulation to safety cues, we tested the necessity of specific IL projections to the basolateral amygdala (BLA) or central amygdala (CeA) during safety recall. Male Long Evans rats were used since prior work showed female Long Evans rats did not acquire the safety discrimination task used in this study. Here, we show the infralimbic projection to the central amygdala was necessary for suppressing fear cue-induced freezing in the presence of a learned safety cue, and the projection to the basolateral amygdala was not. The loss of discriminative fear regulation seen specifically during IL->CeA inhibition is similar to the behavioral disruption seen in PTSD individuals that fail to regulate fear in the presence of a safety cue.

Background: The purpose of this proof-of-concept feasibility study was to determine if spike-triggered intraspinal microstimulation (ISMS), a form of activity dependent stimulation (ADS), results in improved motor performance in an ambulatory rat model of spinal cord injury (SCI). Methods: Experiments were carried out in adult male Sprague Dawley rats with moderate thoracic contusion injury. Rats were assigned to one of two groups: Control or ADS therapy. Four weeks post-SCI, all rats were implanted with a recording microelectrode in the left hindlimb motor cortex and a fine-wire stimulating electrode in the contralateral lumbar spinal cord. ADS was administered for 4 hours/day, 4 days/week, for 4 weeks. During therapy sessions, single-unit spikes were discriminated in real time in the hindlimb motor cortex and used to trigger stimulation in the spinal cord ventral horn. Control rats were similarly implanted with electrodes but did not receive stimulation therapy. Results: Motor performances of each rat were evaluated before SCI contusion, once a week post-SCI for four weeks (prior to electrode implantation), and once a week post-conditioning for four weeks. Basso, Beattie, and Bresnahan (BBB) locomotor scores were significantly improved in ADS rats compared to Control rats at 1 and 2 weeks after initiation of therapy. Foot fault scores on the Horizontal Ladder were significantly improved in ADS rats compared to pre-therapy ADS and Control rats after 1 week of therapy and recovered to near pre-injury scores after 3 weeks of therapy. The Ledged Beam test showed deficits after SCI in both ADS and Control rats but there were no significant differences between groups after 4 weeks of ADS therapy. Conclusions: These results show that chronic stimulation after spinal cord injury using a methodology of spike-triggered ISMS enhances behavioral recovery of locomotor function as measured by the BBB score and the Horizontal Ladder task. However, it is still uncertain if the behavioral improvements seen were dependent on spike-triggered ISMS.

Sensory systems preferentially strengthen responses to stimuli based on their reliability at conveying accurate information. While previous reports demonstrate that the brain reweighs cues based on dynamic changes in reliability, how the brain may learn and maintain neural responses to sensory statistics expected to be stable over time is unknown. The barn owl’s midbrain features a map of auditory space where neurons compute horizontal sound location from the interaural time difference (ITD). Frequency tuning of midbrain map neurons correlates with the most reliable frequencies for the neurons’ preferred ITD (Cazettes et al., 2014). Removal of the facial ruff led to a specific decrease in the reliability of high frequencies from frontal space. To directly test whether permanent changes in ITD reliability drive frequency tuning, midbrain map neurons were recorded from adult owls, with the facial ruff removed during development, and juvenile owls, before facial ruff development. In both groups, frontally tuned neurons were tuned to frequencies lower than in normal adult owls, consistent with the change in ITD reliability. In addition, juvenile owls exhibited more heterogeneous frequency tuning, suggesting normal developmental processes refine tuning to match ITD reliability. These results indicate causality of long-term statistics of spatial cues in the development of midbrain frequency tuning properties, implementing probabilistic coding for sound localization.

Objective. Neuropixels (NP) probes are a significant advance in electrophysiological recording technology that enable monitoring of hundreds of neurons in the brain simultaneously at different depths. Application of this technology has been predominately in rodents, however widespread use in non-human primates (NHPs) such as rhesus macaques has been limited. In this study we sought to overcome two overarching challenges that impede acute NP implantation in NHPs: (1) traditional microdrive systems that mount to cephalic chambers are commonly used to access cortical areas for microelectrode recordings but are not designed to accommodate NP probes, and (2) NHPs have thick dura mater and tissue growth within the cephalic chambers which poses a challenge for insertion of the extremely fragile NP probe. Approach. In this study we present a novel NP guide tube system that can be adapted to commercial microdrive systems and demonstrate an implant method using the NP guide tube system. This system was developed using a combination of CAD design, 3D printing, and small part machining. Software programs, 3D Slicer and SolidWorks were used to target cortical areas, approximate recording depths and locations, and for in-silico implant testing. Main results. We performed in vivo testing to validate our methodology, successfully implanting, explanting, and reimplanting NP probes. We collected stable neurophysiological recordings in the premotor cortex of a rhesus macaque at rest and during performance of a reaching task. Significance. In this study we demonstrate a robust Neuropixels implant system that allows multiple penetrations with the same NP probe and share design files that will facilitate the adoption of this powerful recording technology for NHP studies.

Growing evidence suggests that brain activity during sleep, as well as sleep regulation, are tightly linked with synaptic function and network excitability at the local and global levels. We previously reported that a mutation in synaptobrevin 2 ( Vamp2 ) in restless ( rlss ) mice results in a marked increase of wakefulness and suppression of sleep, in particular REM sleep (REMS) as well as increased consolidation of sleep and wakefulness. In the current study, using finer-scale in vivo electrophysiology recordings, we report that spontaneous cortical activity in rlss mice during NREM sleep (NREMS) is characterised by an occurrence of abnormally prolonged periods of complete neuronal silence (OFF-periods), often lasting several seconds, similar to the burst suppression pattern typically seen under deep anaesthesia. Increased incidence of prolonged network OFF-periods was not specific to NREMS, but also present in REMS and wake in rlss mice. Slow-wave activity (SWA) was generally increased in rlss mice, while higher frequencies including theta-frequency activity were decreased, further resulting in diminished differences between vigilance states. The relative increase in SWA after sleep deprivation was attenuated in rlss mice, suggesting either that rlss mice experience persistently elevated sleep pressure, or, alternatively, that the intrusion of sleep-like patterns of activity into awake state diminishes the accumulation of sleep drive. We propose that deficit in global synaptic neurotransmitter release leads to ‘state inertia’, reflected in an abnormal propensity of brain networks to enter and remain in a persistent ‘default state’ resembling coma or deep anaesthesia.

Sensory cortical areas are robustly modulated by higher-order cortices. Our previous study shows that the anterior cingulate cortex (ACC) can immediately and transiently enhance responses in the mouse auditory cortex (ACx). Here, we further examined whether strong activation of ACC neurons can induce long-term effects in mice of both sexes. To our surprise, only stimulation of cell bodies in the ACC, but not ACC-to-ACx terminal activation, induced long-term enhancement of auditory responses in the ACx. Anatomical examination showed that the ACC indirectly projects to the ACx via the rhinal cortex (RCx). High-frequency stimulation of ACC-projecting terminals to the RCx or RCx-projecting terminals to the ACx induced a similar effect as the cell body activation of ACC neurons, whereas silencing the RCx blocked this long-term enhancement. High-frequency stimulation of ACC projections to the RCx also induced long-term augmentation of sound-evoked flight behavior in male mice. These results show that the ACC promotes the long-term enhancement of auditory responses in the ACx through an indirect pathway via the RCx. SIGNIFICANCE STATEMENT In this study, we demonstrate that the anterior part of the anterior cingulate cortex (ACC) evokes long-term enhancement of auditory responses in the auditory cortex (ACx) when it is strongly activated. Importantly, instead of a direct projection, we show that the ACC implements this effect via an indirect pathway through the lateral rhinal cortex using a series of physiological, optogenetic, anatomic, and behavioral experiments. Along with a short-term effect, this long-term enhancement induced by an indirect ACC-to-ACx projection could increase the odds of survival when animals are faced with threats after a significant event.

Biocompatible and plastic neural interface devices allow for minimally invasive recording of brain activity. Increasing electrode density in such devices is essential for high-resolution neural recordings. Superimposing conductive leads in devices can help multiply the number of recording sites while keeping probes width small and suitable for implantation. However, because of leads' vertical proximity, this can create capacitive coupling (CC) between overlapping channels, which leads to crosstalk. Here, we present a thorough investigation of CC phenomenon in multi-gold layer thin-film multi-electrode arrays with a parylene C (PaC) insulation layer between superimposed leads. We also propose a guideline on the design, fabrication, and characterization of such type of neural interface devices for high spatial resolution recording. Our results demonstrate that the capacitance created through CC between superimposed tracks decreases non-linearly and then linearly with the increase of insulation thickness. We identify an optimal PaC insulation thickness that leads to a drastic reduction of CC between superimposed gold channels while not significantly increasing the overall device thickness. Finally, we show that double gold layer electrocorticography probes with the optimal insulation thickness exhibit similar performances in vivo when compared to single-layer devices. This confirms that these probes are adequate for high-quality neural recordings.

The medial superior olive (MSO) is a binaural nucleus that is specialized in detecting the relative arrival times of sounds at both ears. Excitatory inputs to its neurons originating from either ear are segregated to different dendrites. To study the integration of synaptic inputs both within and between dendrites, we made juxtacellular and whole-cell recordings from the MSO in anesthetized female gerbils, while presenting a “double zwuis” stimulus, in which each ear received its own set of tones, which were chosen in a way that all second-order distortion products (DP2s) could be uniquely identified. MSO neurons phase-locked to multiple tones within the multitone stimulus, and vector strength, a measure for spike phase-locking, generally depended linearly on the size of the average subthreshold response to a tone. Subthreshold responses to tones in one ear depended little on the presence of sound in the other ear, suggesting that inputs from different ears sum linearly without a substantial role for somatic inhibition. The “double zwuis” stimulus also evoked response components in the MSO neuron that were phase-locked to DP2s. Bidendritic subthreshold DP2s were quite rare compared with bidendritic suprathreshold DP2s. We observed that in a small subset of cells, the ability to trigger spikes differed substantially between both ears, which might be explained by a dendritic axonal origin. Some neurons that were driven monaurally by only one of the two ears nevertheless showed decent binaural tuning. We conclude that MSO neurons are remarkably good in finding binaural coincidences even among uncorrelated inputs. SIGNIFICANCE STATEMENT Neurons in the medial superior olive are essential for precisely localizing low-frequency sounds in the horizontal plane. From their soma, only two dendrites emerge, which are innervated by inputs originating from different ears. Using a new sound stimulus, we studied the integration of inputs both within and between these dendrites in unprecedented detail. We found evidence that inputs from different dendrites add linearly at the soma, but that small increases in somatic potentials could lead to large increases in the probability of generating a spike. This basic scheme allowed the MSO neurons to detect the relative arrival time of inputs at both dendrites remarkably efficient, although the relative size of these inputs could differ considerably.

Wireless brain technologies are empowering basic neuroscience and clinical neurology by offering new platforms that minimize invasiveness and refine possibilities during electrophysiological recording and stimulation. Despite their advantages, most systems require on-board power supply and sizeable transmission circuitry, enforcing a lower bound for miniaturization. Designing new minimalistic architectures that can efficiently sense neurophysiological events will open the door to standalone microscale sensors and minimally invasive delivery of multiple sensors. Here we present a circuit for sensing ionic fluctuations in the brain by an ion-sensitive field effect transistor that detunes a single radiofrequency resonator in parallel. We establish sensitivity of the sensor by electromagnetic analysis and quantify response to ionic fluctuations in vitro. We validate this new architecture in vivo during hindpaw stimulation in rodents and verify correlation with local field potential recordings. This new approach can be implemented as an integrated circuit for wireless in situ recording of brain electrophysiology.

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

We replicated behaviorally relevant temporal patterns of synaptic activity in vitro and used the same patterns during sensory stimulation in vivo. There was a Hebbian spike timing-dependent plasticity (STDP) pattern in vitro, but sensory responses in vivo did not shift according to STDP predictions. Analysis suggests that this disparity is influenced by differences in polysynaptic activity, including inhibitory interneurons. These results suggest that STDP rules at synapses in vitro do not necessarily apply to circuits in vivo.

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.