Adaptation of Microelectrode Array Technology for the Study of Anesthesia-Induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain. This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN

MAPPING LOW-FREQUENCY FIELD POTENTIALS IN BRAIN CIRCUITS WITH HIGH-RESOLUTION CMOS ELECTRODE ARRAY RECORDINGS

Neurotechnologies based on microelectronic active electrode array devices are on the way to provide the capability to record electrophysiological neural activity from a thousands of closely spaced microelectrodes. This generates increasing volumes of experimental data to be analyzed, but also offers the unprecedented opportunity to observe bioelectrical signals at high spatial and temporal resolutions in large portions of brain circuits. The overall aim of this PhD was to study the application of high-resolution CMOS-based electrode arrays (CMOS-MEAs) for electrophysiological experiments and to investigate computational methods adapted to the analysis of the electrophysiological data generated by these devices. A large part of my work was carried out on cortico-hippocampal brain slices by focusing on the hippocampal circuit. In the history of neuroscience, a major technological advance for hippocampal research, and also for the field of neurobiology, was the development of the in vitro hippocampal slice preparation. Neurobiological principles that have been discovered from work on in vitro hippocampal preparations include, for instance, the identification of excitatory and inhibitory synapses and their localization, the characterization of transmitters and receptors, the discovery of long-term potentiation (LTP) and long-term depression (LTD) and the study of oscillations in neuronal networks. In this context, an initial aim of my work was to optimize the preparation and maintenance of acute cortico-hippocampal brain slices on planar CMOS-MEAs. At first, I focused on experimental methods and computational data analysis tools for drug-screening applications based on LTP quantifications. Although the majority of standard protocols still use two electrodes platforms for quantifying LTP, in my PhD I investigate the potential advantages of recording the electrical activity from many electrodes to spatiotemporally characterized electrically induced responses. This work also involved the collaboration with 3Brain AG and a CRO involved in drug-testing, and led to a software tool that was licensed for developing its exploitation. In a second part of my work I focused on exploiting the recording resolution of planar CMOS-MEAs to study the generation of sharp wave ripples (SPW-Rs) in the hippocampal circuit. This research activity was carried out also by visiting the laboratory of Prof. A. Sirota (Ludwig Maximilians University, Munich). In addition to set-up the experimental conditions to record SPW-Rs from planar CMOS-MEAs integrating 4096 microelectrodes, I also explored the implementation of a data analysis pipeline to identify spatiotemporal features that might characterize different type of in-vitro generated SPW-R events. Finally, I also contributed to the initial implementation of high-density implantable CMOS-probes for in-vivo electrophysiology with the aim of evaluating in vivo the algorithms that I developed and investigated on brain slices. With this aim, in the last period of my PhD I worked on the development of a Graphical User Interface for controlling active dense CMOS probes (or SiNAPS probes) under development in our laboratory. I participated to preliminary experimental recordings using 4-shank CMOS-probes featuring 1024 simultaneously recording electrodes and I contributed to the development of a software interface for executing these experiments

CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS

An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus. To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria. Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time

Assessing functional activity of astrocytes by calcium imaging: how do astrocytes respond to the electrophysiological microenvironment

Apesar de não serem capazes de produzir potenciais de acção, é sabido que os astrócitos integram as sinapses, sendo capazes de detectar e responder a estímulos externos com dinâmicas de cálcio espaciotemporalmente complexas, podendo modelar a transmissão sináptica. O objectivo deste projecto é avaliar as dinâmincas de cálcio dos astrócitos através da modelação do seu microambiente electrofisiológico. Para tal, culturas de astrócitos foram estimuladas recorrendo a ThinMEAs©, monitorizando a actividade de cálcio. Os resultados obtidos demonstraram que os astrócitos respondem a estímulos de ±600mV ou ±800mV, gerando uma onda de cálcio que se propaga para células vizinhas. A amplitude, tempo de subida e velocidade de propagação da onda de cálcio está dependente do estímulo, sendo que um estímulo de maior amplitude resulta numa resposta de maior amplitude, demorando mais tempo a atingir o seu pico máximo mas atingindo distâncias mais longas. Apesar de preliminares, estes resultados indicam que os astrócitos são capazes de detectar e responder a mudanças eléctricas externas. Desta forma, os astrócitos são células electricamente excitáveis, possivelmente através do seguinte mecanismo: a estimulação leva à abertura dos canais de cálcio voltagem-dependentes de maneira dependente da voltagem, que irá sensibilizar o retículo endoplasmático resultando numa cascata de libertação de cálcio, gerando uma onda de cálcio que se irá propagar através de junções comunicantes ou gliotransmissão vesicular.Although not able to generate action potentials, it is known that astrocytes integrate synapses, being able to sense and respond to external stimuli with complex calcium dynamics, having the ability to shape synaptic transmission. The aim of this project is to assess astrocytic calcium dynamics upon the modulation of their eletrophysiological microenvironment. To accomplish this, astrocyte cultures were electrically stimulated using ThinMEAs© while monitoring their calcium activity. Obtained data showed that astocytes respond to a ±600mV or ±800mV stimulus by generating a calcium wave which propagates to neighboring cells. The amplitude, rise time and propagation velocity of the calcium wave is dependent on the stimulus, with a higher stimulation amplitude leading to a higher response amplitude, wich takes longer to reach its maximum peak but reach a larger distance. Although preliminary, these results indicate that astrocytes are able to sense and respond to changes of the electrical environment. In this way, astrocytes are electrically excitable cells, possibly due to the following mechanism: electrical stimulation causes voltage-gated calcium channels to open in a voltage-dependent manner, which will sensitize the endoplasmic reticulum leading to a cascade of calcium releases, generating a calcium wave, which will propagate through gap junctions or vesicular gliotransmission

Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca²⁺ Imaging: A Practical Guide to Experiments and "Big Data" Analysis.

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca <sup>2+</sup> imaging experiments in neurons, they are less useful for analyzing Ca <sup>2+</sup> dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca <sup>2+</sup> transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: <i>MultiROI_TZ_profiler</i> for interactive graphing of several movable ROIs simultaneously, <i>Gaussian_Filter5D</i> for Gaussian filtering in several dimensions, and <i>Correlation_Calculator</i> for computing various cross-correlation parameters on voxel collections through time

Accelerating the development of implantable neurochemical biosensors by using existing clinically applied depth electrodes

In this study, an implantable stereo-electroencephalography (sEEG) depth electrode was functionalised with an enzyme coating for enzyme-based biosensing of glucose and L-glutamate. This was done because personalised medicine could benefit from active real-time neurochemical monitoring on small spatial and temporal scales to further understand and treat neurological disorders. To achieve this, the sEEG depth electrode was characterised using cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) using several electrochemical redox mediators (potassium ferri/ferrocyanide, ruthenium hexamine chloride, and dopamine). To improve performance, the Pt sensors on the sEEG depth electrode were coated with platinum black and a crosslinked gelatin-enzyme film to enable enzymatic biosensing. This characterisation work showed that producing a useable electrode with a good electrochemical response showing the expected behaviour for a platinum electrode was possible. Coating with Pt black improved the sensitivity to H2O2 over unmodified electrodes and approached that of well-defined Pt macro disc electrodes. Measured current showed good dependence on concentration, and the calibration curves report good sensitivity of 29.65 nA/cm2/μM for glucose and 8.05 nA/cm2/μM for L-glutamate with a stable, repeatable, and linear response. These findings demonstrate that existing clinical electrode devices can be adapted for combined electrochemical and electrophysiological measurement in patients and obviate the need to develop new electrodes when existing clinically approved devices and the associated knowledge can be reused. This accelerates the time to use and application of in vivo and wearable biosensing for diagnosis, treatment, and personalised medicine

In vivo microelectrode arrays for detecting multi-region epileptic activities in the hippocampus in the latent period of rat model of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a form of refractory focal epilepsy, which includes a latent period and a chronic period. Microelectrode arrays capable of multi-region detection of neural activities are important for accurately identifying the epileptic focus and pathogenesis mechanism in the latent period of TLE. Here, we fabricated multi-shank MEAs to detect neural activities in the DG, hilus, CA3, and CA1 in the TLE rat model. In the latent period in TLE rats, seizures were induced and changes in neural activities were detected. The results showed that induced seizures spread from the hilus and CA3 to other areas. Furthermore, interneurons in the hilus and CA3 were more excited than principal cells and exhibited rhythmic oscillations at approximately 15 Hz in grand mal seizures. In addition, the power spectral density (PSD) of neural spikes and local field potentials (LFPs) were synchronized in the frequency domain of the alpha band (9–15 Hz) after the induction of seizures. The results suggest that fabricated MEAs have the advantages of simultaneous and precise detection of neural activities in multiple subregions of the hippocampus. Our MEAs promote the study of cellular mechanisms of TLE during the latent period, which provides an important basis for the diagnosis of the lesion focus of TLE

In vitro neuronal cultures on MEA: an engineering approach to study physiological and pathological brain networks

Reti neuronali accoppiate a matrici di microelettrodi: un metodo ingegneristico per studiare reti cerebrali in situazioni fisiologiche e patologich

Reducing the Societal Costs of Traumatic Brain Injury: Astrocyte-Based Therapeutics and Functional Injury Tolerance of the Living Brain

Approximately 1.7 million traumatic brain injuries (TBI) occur annually in the United States, with an annual estimated societal cost of at least $76.5 billion. Addressing the growing TBI epidemic will require a multi pronged approach: developing novel treatment strategies and enhancing existing preventative measures. The specific aims of this thesis are: (1) to modulate astrocyte activation as a potential therapeutic strategy post TBI, (2) to determine the relationship between tissue deformation and alterations in electrophysiological function in the living brain, and (3) to investigate underlying mechanisms of functional changes post TBI by utilizing stretchable microelectrode arrays (SMEAs). In response to disease or injury, astrocytes become activated in a process called reactive astrogliosis. Activated astrocytes generate harmful radicals that exacerbate brain damage and can hinder regeneration of damaged neural circuits by secreting neuro developmental inhibitors and glycosaminoglycans (GAGs). Since mechanically-activated astrocytes upregulate GAG production, delivery of GFP-TAT, a mock therapeutic protein conjugated to the cell-penetrating peptide TAT, increased significantly after activation. A TAT-conjugated peptide JNK inhibitor was delivered to activated astrocytes and significantly reduced activation. These results suggest a potentially new, targeted therapeutic utilizing TAT for preventing astrocyte activation with the possibility of limiting off-target, negative side effects. While modulating astrocyte activation is a promising treatment strategy for TBI, effective therapeutic treatments are still lacking. Preventing TBI, by developing more effective safety systems, remains crucial. We determined functional tolerance criteria for the hippocampus and cortex based on alterations in electrophysiological function in response to controlled mechanical stimuli. Organotypic hippocampal and cortical slice cultures were mechanically injured at tissue strains and strain rates relevant to TBI, and changes in electrophysiological function were quantified. Most changes in electrophysiological function were dependent on strain and strain rate in a complex, nonlinear manner. Our results provide functional data that can be incorporated into finite element (FE) models to improve their biofidelity of accident and collision reconstructions. TBI causes alterations in macroscopic function and behavior, which can be characterized by alterations in electrophysiological function in vitro. We utilized a novel in vitro platform for TBI research, the SMEA, to investigate the effects of TBI on pharmacologically induced, long lasting network synchronization in the hippocampus. Mechanical stimulation of organotypic hippocampal slice cultures significantly disrupted this network synchronization 24 hours after injury. Our results suggest that the ability of the hippocampal neuronal network to develop and sustain network synchronization was disrupted after mechanical injury, while also demonstrating the utility of the SMEA for TBI research. Herein, we identified a novel therapeutic strategy for treating the deleterious effects of astrocyte activation post-TBI. We also developed tolerance criteria relating mechanical injury parameters to electrophysiological function, an important step in developing more accurate computational simulations of TBI. Equipping FE models with new information on the functional response of the living brain will enhance their biofidelity, potentially leading to improved safety systems while reducing development costs. Finally, we utilized a novel in vitro TBI research platform, the SMEA, to investigate the effects of TBI on long-lasting network synchronization in the hippocampus. Compared to more labor intensive in vivo approaches, the ability of the SMEA to efficiently test TBI hypotheses within a single organotypic slice culture over extended durations could increase the speed of drug discovery through high-content screening. This multi-pronged approach is necessary to address the growing public health concern of TBI

Developing novel biosensors for detection of L-aspartate and N-acetyl aspartate

Microelectrode biosensors have proven to be an invaluable tool in direct measurement of extracellular concentration of analytes. Real-time measurement of released neuroactive compounds with simultaneous recording of electrophysiological activity has been made possible because of enzyme based sensors specific for their analytes. By using the sol-gel coating method of sensor fabrication, enzyme-based sensors for L-aspartate (L-Asp) and N-acetyl aspartate (NAA) were fabricated on a Platinum (Pt) microelectrode. The L-Asp sensor was made using the enzyme L-aspartate oxidase (LAO) and was fully characterised. The sensor achieved a steady state response to L-Asp within 15 s, had a lower detection limit of 3 μM with a sensitivity of 0.0016 μA/μM/cm2 (R=0.99). The L-Asp sensor also detected real-time release of L-Asp in area CA1 of hippocampal brain slices. L-Asp release was enhanced by independent application of excitatory amino acid transporter blocker DL-threo-β-Benzyloxyasparticacid (TBOA) and L-albizziine (L-Alb) an inhibitor for L-Asp metabolising enzyme. L-Alb also induced seizure-like activity with accompanying L-Asp release. These results bolster the idea of L-Asp as a possible co-transmitter or neuromodulator. NAA is a derivative of L-Asp and is the second most abundant compound in the brain after L-glutamate. NAA is also a marker for stroke and traumatic brain injury. NAA sensor was fabricated using the enzymes aspartoacylase (ASPA) and LAO. A novel method for detection of ASPA activity and proof of concept data for the NAA sensor is presented. The principle and methodology for NAA sensor fabrication can be extended to develop integrated electrochemical sensor array such as SMARTChip. Both the L-Asp and NAA sensor can provide valuable insight into the role of these compounds in functional aspects of brain physiology