low-cost-electroencephalographic-recording-system-combined-with

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain <em>In Vivo</em>

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Hokkaido University

To improve the performance of cochlear implants, we have integrated a microdevice into a model of the auditory periphery with the goal of creating a microprocessor. We constructed an artificial peripheral auditory system using a hybrid model in which polyvinylidene difluoride was used as a piezoelectric sensor to convert mechanical stimuli into electric signals. To produce frequency selectivity, the slit on a stainless steel base plate was designed such that the local resonance frequency of the membrane over the slit reflected the transfer function. In the acoustic sensor, electric signals were generated based on the piezoelectric effect from local stress in the membrane. The electrodes on the resonating plate produced relatively large electric output signals. The signals were fed into a computer model that mimicked some functions of inner hair cells, inner hair cell-auditory nerve synapses, and auditory nerve fibers. In general, the responses of the model to pure-tone burst and complex stimuli accurately represented the discharge rates of high-spontaneous-rate auditory nerve fibers across a range of frequencies greater than 1 kHz and middle to high sound pressure levels. Thus, the model provides a tool to understand information processing in the peripheral auditory system and a basic design for connecting artificial acoustic sensors to the peripheral auditory nervous system. Finally, we discuss the need for stimulus control with an appropriate model of the auditory periphery based on auditory brainstem responses that were electrically evoked by different temporal pulse patterns with the same pulse number. 

A hardware model of the auditory periphery to transduce acoustic signals into neural activity.

In this report, we describe the system integration of a complementary metal oxide semiconductor (CMOS) integrated circuit (IC) chip, capable of both stimulation and recording of neurons or neural tissues, to investigate electrical signal propagation within cellular networks in vitro. The overall system consisted of three major subunits: a 5.0 × 5.0 mm CMOS IC chip, a reconfigurable logic device (field-programmable gate array, FPGA), and a PC. To test the system, microelectrode arrays (MEAs) were used to extracellularly measure the activity of cultured rat cortical neurons and mouse cortical slices. The MEA had 64 bidirectional (stimulation and recording) electrodes. In addition, the CMOS IC chip was equipped with dedicated analog filters, amplification stages, and a stimulation buffer. Signals from the electrodes were sampled at 15.6 kHz with 16-bit resolution. The measured input-referred circuitry noise was 10.1 μ V root mean square (10 Hz to 100 kHz), which allowed reliable detection of neural signals ranging from several millivolts down to approximately 33 μ Vpp. Experiments were performed involving the stimulation of neurons with several spatiotemporal patterns and the recording of the triggered activity. An advantage over current MEAs, as demonstrated by our experiments, includes the ability to stimulate (voltage stimulation, 5-bit resolution) spatiotemporal patterns in arbitrary subsets of electrodes. Furthermore, the fast stimulation reset mechanism allowed us to record neuronal signals from a stimulating electrode around 3 ms after stimulation. We demonstrate that the system can be directly applied to, for example, auditory neural prostheses in conjunction with an acoustic sensor and a sound processing system.

A CMOS IC-based multisite measuring system for stimulation and recording in neural preparations in vitro.

Anesthesia affects central auditory processing. However, it is unclear to what extent the choice of anesthetic agent affects neural responses to sound stimulation. A mixture of three anesthetics (medetomidine, midazolam and butorphanol; MMB) was recently developed as an alternative to ketamine owing to the latter's addictive potential, yet the effect of this combination of anesthetics on neural responses is not known. Here, we compared the spontaneous activity, tuning properties and temporal responses of primary auditory cortical neurons under these two anesthetic conditions, using electrophysiological and flavoprotein autofluorescence imaging methods. Frequency tuning properties were not significantly different between ketamine and MMB anesthesia. However, neural activity under MMB showed decreases in the spontaneous and tone-evoked firing rates in a layer-dependent manner. Moreover, the temporal response patterns were also different between the anesthetics in a layer-dependent manner, which may reflect differences in the anesthetic mechanisms. These results demonstrated how response properties in the primary auditory cortex are affected by the choice of anesthesia.

Neural response differences in the rat primary auditory cortex under anesthesia with ketamine versus the mixture of medetomidine, midazolam and butorphanol.

The effects of anesthesia on the functional auditory characteristics of cortical neurons, such as spatial and temporal response properties, vary between an anesthetized and an awake subject. However, studies have shown that an appropriate anesthetic method that approaches the awake condition is still useful because of its greater stability and controllability. The present study compared neural response properties from two core fields of the mouse auditory cortex under three anesthetic conditions: urethane; ketamine and xylazine hydrochloride (KX) mixture; and a combination of medetomidine, midazolam, and butorphanol (MMB). To measure sound stimulation in vivo, we recorded flavoprotein-autofluorescent images of endogenous green fluorescence. Under all conditions, fluorescence changes in auditory core subfields in response to tones were observed, and response properties, such as peak intensity, latency, duration, and activated areas were analyzed. Results showed larger response peak intensity, latency, and duration in the core subfields under urethane compared with KX and MMB, with no significant differences between KX and MMB. Conversely, under KX anesthesia the activated areas showed characteristic response properties in a subfield-dependent manner. These results demonstrated the varied effects of anesthesia on response properties in the core subfields of the auditory cortex.

Transcranial flavoprotein-autofluorescence imaging of sound-evoked responses in the mouse auditory cortex under three types of anesthesia.

To examine local network properties of the mouse auditory cortex in vitro, we recorded extracellular spatiotemporal laminar profiles driven by short electric local stimulation on a planar multielectrode array substrate. The recorded local field potentials were subsequently evaluated using current source density (CSD) analysis to identify sources and sinks. Current sinks are thought to be an indicator of net synaptic current in the small volume of cortex surrounding the recording site. Thus, CSD analysis combined with multielectrode arrays enabled us to compare mean synaptic activity in response to small current stimuli on a layer-by-layer basis. We also used senescence-accelerated mice (SAM), some strains of which show earlier onset of age-related hearing loss, to examine the characteristic spatiotemporal CSD profiles stimulated by electrodes in specific cortical layers. Thus, the CSD patterns were classified into several clusters based on stimulation sites in the cortical layers. We also found some differences in CSD patterns between the two SAM strains in terms of aging according to principle component analysis with dimension reduction. For simultaneous two-site stimulation, we modeled the obtained CSD profiles as a linear superposition of the CSD profiles to individual single-site stimulation. The model analysis indicated the nonlinearity of spatiotemporal integration over stimulus-driven activity in a layer-specific manner. Finally, on the basis of these results, we discuss the auditory cortex local network properties and the effects of aging on these mouse strains.

An analysis of current source density profiles activated by local stimulation in the mouse auditory cortex in vitro.

Salicylate is the active ingredient in aspirin, and in high-doses it is used as an experimental tool to induce transient hearing loss, tinnitus, and hyperacusis. These salicylate-induced perceptual disturbances are associated with tonotopic-map reorganization and neural activity modulation, and such neural correlates have been examined in the central auditory pathway, including the auditory cortex (AC). Although previous studies have reported that salicylate induces increases in noise-burst-evoked neural responses and reorganization of tonotopic maps in the primary AC, little is known about the effects of salicylate on other frequency-organized AC subfields such as the anterior auditory, secondary auditory, and dorsomedial fields. Therefore, to examine salicylate-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of sodium salicylate (SS, 200 mg/kg), using flavoprotein auto-fluorescence imaging. SS-treatment gradually reduced responses driven by tone-bursts with lower (≤8 kHz) and higher (≥25 kHz) frequencies over 3 h, whereas evoked responses to tone-bursts within middle-range frequencies (e.g., 12 and 16 kHz) were sustained and unchanged in the four subfields. Additionally, in each of the four subfields, SS-treatment induced similar reorganization of tonotopic maps, and the response areas selectively driven by the middle-range frequencies were profoundly expanded. Our results indicate that the SS-induced tonotopic map reorganizations in each of the four AC subfields were similar, and only the extent of the activated areas responsive to tone-bursts with specific frequencies was subfield-dependent. Thus, we expect that examining cortical reorganization induced by SS may open the possibility of new treatments aimed at altering cortical reorganization into the normative functional organization.

Salicylate-induced frequency-map reorganization in four subfields of the mouse auditory cortex.

Chronic neural recording in freely moving animals is important for understanding neural activities of cortical neurons associated with various behavioral contexts. In small animals such as mice, it has been difficult to implant recording electrodes into exact locations according to stereotactic coordinates, skull geometry, or the shape of blood vessels. The main reason for this difficulty is large individual differences in the exact location of the targeted brain area.

Flavoprotein fluorescence imaging-based electrode implantation for subfield-targeted chronic recording in the mouse auditory cortex.

Magnetic stimulation is widely used in neuroscience research and clinical treatment. Despite recent progress in understanding the neural modulation mechanism of conventional magnetic stimulation methods, the physiological mechanism at the cortical microcircuit level is not well understood due to the poor stimulation focality and large electric artifact in the recording. To overcome these issues, we used a sub-millimeter-sized coil (micro-coil) to stimulate the mouse auditory cortex in vivo. To determine the mechanism, we conducted the first direct electrophysiological recording of micro-coil-driven neural responses at multiple sites on the horizontal surface and laminar areas of the auditory cortex. The laminar responses of local field potentials (LFPs) to the magnetic stimulation reached layer 6, and the spatiotemporal profiles were very similar to those of the acoustic stimulation, suggesting the activation of the same cortical microcircuit. The horizontal LFP responses to the magnetic stimulation were evoked within a millimeter-wide area around the stimulation coil. The activated cortical area was dependent on the coil orientation, providing useful information on the effective position of the coil relative to the brain surface for modulating cortical circuitry activity. In addition, numerical calculation of the induced electric field in the brain revealed that the inhomogeneity of the horizontal electric field to the surface is critical for micro-coil-induced cortical activation. The results suggest that our micro-coil technique has the potential to be used as a chronic, less-invasive and highly focal neuro-stimulator, and is useful for investigating microcircuit responses to magnetic stimulation for clinical treatment.

Micro-coil-induced Inhomogeneous Electric Field Produces sound-driven-like Neural Responses in Microcircuits of the Mouse Auditory Cortex In Vivo.

The prevalence of tinnitus is known to increase with age. The age-dependent mechanisms of tinnitus may have important implications for the development of new therapeutic treatments. High doses of salicylate can be used experimentally to induce transient tinnitus and hearing loss. Although accumulating evidence indicates that salicylate induces tinnitus by directly targeting neurons in the peripheral and central auditory systems, the precise effect of salicylate on neural networks in the auditory cortex (AC) is unknown. Here, we examined salicylate-induced changes in stimulus-driven laminar responses of AC slices with salicylate superfusion in young and aged senescence-accelerated-prone (SAMP) and -resistant (SAMR) mice. Of the two strains, SAMP1 is known to be a more suitable model of presbycusis. We recorded stimulus-driven laminar local field potential (LFP) responses at multi sites in AC slice preparations. We found that for all AC slices in the two strains, salicylate always reduced stimulus-driven LFP responses in all layers. However, for the amplitudes of the LFP responses, the two senescence-accelerated mice (SAM) strains showed different laminar properties between the pre- and post-salicylate conditions, reflecting strain-related differences in local circuits. As for the relationships between auditory brainstem response (ABR) thresholds and the LFP amplitude ratios in the pre- vs. post-salicylate condition, we found negative correlations in layers 2/3 and 4 for both older strains, and in layer 5 (L5) in older SAMR1. In contrast, the GABAergic agonist muscimol (MSC) led to positive correlations between ABR thresholds and LFP amplitude ratios in the pre- vs. post-MSC condition in younger SAM mice from both strains. Further, in younger mice, salicylate decreased the firing rate in AC L4 pyramidal neurons. Thus, salicylate can directly reduce neural excitability of L4 pyramidal neurons and thereby influence AC neural circuit activity. That we observed age-dependent effects of salicylate and varied GABAergic sensitivity in the AC among mouse strains with hearing loss implies that potential therapeutic mechanisms for tinnitus may operate differently in young vs. aged subjects. Therefore, scientists developing new therapeutic modalities for tinnitus treatment should consider using both aged and young animals.

Salicylate-Induced Suppression of Electrically Driven Activity in Brain Slices from the Auditory Cortex of Aging Mice.

Ototoxic-drug-induced hearing disturbances in the auditory periphery are associated with tonotopic map reorganization and neural activity modulation, as well as changes in neural correlates in the central auditory pathway, including the auditory cortex (AC). Previous studies have reported that peripheral auditory impairment induces AC plasticity that involves changes in the balance of excitatory vs. inhibitory synapses, within existing and newly forming patterns of connectivity. Although we know that such plastic changes modulate sound-evoked neural responses and the organization of tonotopic maps in the primary AC (A1), little is known about the effects of peripheral impairment on other frequency-organized AC subfields, such as the anterior auditory field (AAF) and the secondary auditory cortex (A2). Therefore, to examine ototoxic-drug-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of kanamycin sulfate (1 mg/g body weight) and bumetanide (0.05 mg/g body weight), using in vivo transcranial flavoprotein autofluorescence imaging over a 4-week period. At first, ototoxic treatment gradually reduced responses driven by tone bursts with lower- (≤8 kHz) and middle- (e.g., 16 kHz) range frequencies in all AC subfields. Subsequently, response intensities in the A1 recovered to more than 78% of the pre-drug condition; however, in the AAF and A2, they remained significantly lower and were unchanged over 3 weeks. Furthermore, after drug administration, the best frequency (BF) areas of the lower (4 and 8 kHz) and higher (25 and 32 kHz) ranges in all subfields were reduced and shifted to those of a middle range (centered around 16 kHz) during the 3 weeks following drug administration. Our results also indicated that, compared with A1, BF distributions in the AAF and A2 were sharper around 16 kHz 3 weeks after drug administration. These results indicate that the ototoxic-damage-induced tonotopic map reorganizations that occurred in each of the three AC subfields were similar, but that there were subfield-dependent differences in the extent of response intensities and in the activated areas that were responsive to tone bursts with specific frequencies. Thus, by examining cortical reorganization induced by ototoxic drugs, we may contribute to the understanding of how this reorganization can be caused by peripheral damage.

In vivo transcranial flavoprotein autofluorescence imaging of tonotopic map reorganization in the mouse auditory cortex with impaired auditory periphery.

The local application of electrical currents to the cortex is one of the most commonly used techniques to activate neurons, and this intracortical stimulation (ICS) could potentially lead to new types of neuroprosthetic devices that can be directly applied to the cortex. To identify whether ICS-activated circuits are physiological vs. profoundly artificial, it is necessary to record in vivo the responses of the same neuronal population to both natural sensory stimuli and artificial electric stimuli. However, few studies have extensively reported simultaneous electrophysiological recordings combined with ICS. Here, we evaluated the similarity between sound- and ICS-driven cortical response patterns in different cortical layers. In the mouse auditory cortex, we performed laminar recordings using 16-channel silicon electrodes and ICS using sharp glass-pipette electrodes containing biocytin for layer identification. In different cortical depths, short current pulses were delivered in vivo to mice under urethane anesthesia. For the recorded data, we mainly analyzed properties of local field potentials and current source densities (CSDs). We demonstrated that electrical stimulation evoked different excitation patterns according to the stimulated cortical layer; responses to electric stimuli in layer 4 were most likely to mimic acoustic responses. Next, we proposed a CSD-based stimulation method to artificially synthesize sound-driven responses, using an approximation method associated with a linear combination of CSD patterns electrically stimulated in the different cortical layers. The result indicates that synthesized responses were consistent with the canonical model of sound processing. Using these approaches, we provide a new technique in which natural sound-driven responses can be mimicked by well-designed computational stimulation pattern sequences in a layer-dependent manner. These findings may aid in the future development of an electrical stimulation methodology for a cortical prosthesis.

Sound- and current-driven laminar profiles and their application method mimicking acoustic responses in the mouse auditory cortex in vivo.

Single coil-based systems for magnetic stimulation are widely used for neurostimulation in neuroscience research and clinical treatment of neurological diseases. However, parallelization of magnetic stimulation with multiple coils may generate far greater potential than a single coil, and could thus expand the scope of brain area stimulation. Therefore, we examined whether a multiple coil-based system could improve the effectiveness and focality of conventional single coil-based magnetic stimulation.

A multichannel magnetic stimulation system using submillimeter-sized coils: system development and experimental application to rodent brain in vivo.

Ultrasound stimulation is expected to be useful for transcranial local and deep stimulation of the brain, which is difficult to achieve using conventional electromagnetic stimulation methods. Previous ultrasound stimulation experiments have used various types of acute  preparations, including hippocampus slices from rodents and Caenorhabditis elegans tissue. For  preparations, researchers have used the cortices of rodents as targets for transcranial ultrasound stimulation. However, no previous studies have used  ultrasound stimulation in rodent cortical slices to examine the mechanisms of ultrasound-driven central neural circuits. Here we demonstrate the optimal experimental conditions for an  ultrasound stimulation system for measuring activity in brain slices using a multielectrode array substrate. We found that the peak amplitudes of the ultrasound-evoked cortical responses in the brain slices depend on the intensities and durations of the ultrasound stimulation parameters. Thus, our findings provide a new  experimental setup that enables activation of a brain slice  ultrasound stimulation. Accordingly, our results indicate that choosing the appropriate ultrasound waveguide structure and stimulation parameters is important for producing the desired intensity distribution in a localized area within a brain slice. We expect that this experimental setup will facilitate future exploration of the mechanisms of ultrasound-driven neural activity.

A Multielectrode Array-Based Recording System for Analyzing Ultrasound-Driven Neural Responses in Brain Slices .

Transcranial magnetic stimulation (TMS), a minimally/non-invasive method of electromagnetic stimulation of brain tissue, has been shown to be beneficial in clinical therapy for specific neurological diseases and disorders. Magnetic stimulation is also used to modulate human and animal brain activity in basic neuroscience studies. Among experimental animal models, mouse models are particularly popular and uniquely representative of brain disorders in basic neuroscience research. TMS in mouse models may play a substantial role in understanding TMS-induced changes in neural networks and plasticity. Although TMS techniques are widely used to examine rodent disease models, techniques specific for mice using small magnetic stimulators have not been intensively developed. Here, we provide a numerical simulation and a practical method of applying TMS to mice by constructing millimeter-sized TMS coils to deliver a low stimulation intensity while maintaining focality. Our results indicate the TMS coils can produce an electrical field with sufficient magnitude to activate the anesthetized mouse cortex in the presence or absence of the skull in vivo. Our results also show that, immediately after magnetic stimulation, local field and action potentials were reliably observed in a manner that depended on the distance between the coil and the brain, implying even a small coil could reliably evoke cortical activity. Therefore, our results show our millimeter-sized coils could produce electric fields sufficient to alter cortical excitability in mice. These coils could be useful in future preclinical studies to examine detailed mechanisms underlying TMS-induced changes in neural activity of the auditory cortex and other cortical regions.

Article	Year
A hardware model of the auditory periphery to transduce acoustic signals into neural activity. Frontiers in neuroengineering\| PubMed ID: 24324432	2013
A CMOS IC-based multisite measuring system for stimulation and recording in neural preparations in vitro. Frontiers in neuroengineering\| PubMed ID: 25346683	2014
Neural response differences in the rat primary auditory cortex under anesthesia with ketamine versus the mixture of medetomidine, midazolam and butorphanol. Hearing research\| PubMed ID: 27321205	2016
Transcranial flavoprotein-autofluorescence imaging of sound-evoked responses in the mouse auditory cortex under three types of anesthesia. Neuroscience letters\| PubMed ID: 27641319	2016
An analysis of current source density profiles activated by local stimulation in the mouse auditory cortex in vitro. Brain research\| PubMed ID: 28119054	2017
Salicylate-induced frequency-map reorganization in four subfields of the mouse auditory cortex. Hearing research\| PubMed ID: 28637591	2017
Flavoprotein fluorescence imaging-based electrode implantation for subfield-targeted chronic recording in the mouse auditory cortex. Journal of neuroscience methods\| PubMed ID: 28851513	2018
Micro-coil-induced Inhomogeneous Electric Field Produces sound-driven-like Neural Responses in Microcircuits of the Mouse Auditory Cortex In Vivo. Neuroscience\| PubMed ID: 29246784	2018
Salicylate-Induced Suppression of Electrically Driven Activity in Brain Slices from the Auditory Cortex of Aging Mice. Frontiers in aging neuroscience\| PubMed ID: 29311894	2017
In vivo transcranial flavoprotein autofluorescence imaging of tonotopic map reorganization in the mouse auditory cortex with impaired auditory periphery. Hearing research\| PubMed ID: 30981948	2019
Sound- and current-driven laminar profiles and their application method mimicking acoustic responses in the mouse auditory cortex in vivo. Brain research\| PubMed ID: 31323198	2019
A multichannel magnetic stimulation system using submillimeter-sized coils: system development and experimental application to rodent brain in vivo. Journal of neural engineering\| PubMed ID: 31642445	2019
A Multielectrode Array-Based Recording System for Analyzing Ultrasound-Driven Neural Responses in Brain Slices . Frontiers in neuroscience\| PubMed ID: 35273476	2022
Temporal and spatial profiles of evoked activity induced by magnetic stimulation using millimeter-sized coils in the mouse auditory cortex in vivo. Brain research\| PubMed ID: 36115587	2022

Takashi Tateno

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