Versatile LCP surface microelectrodes for combining electrophysiology and in vivo two-photon imaging in the murine CNS

Neurons and astrocytes are highly interconnected and form a complex cellular network for signal processing in the brain. The electrical activity of neurons and astroglial Ca2+ signals are tightly coupled. Parallel recording of electrical activity and Ca2+ signals can help to identify the molecular mechanisms of neuron-glia communications. In this work, flexible liquid crystal polymer microelectrode arrays for electrical recordings and stimulations during two-photon laser-scanning microscopy (2P-LSM) were developed. The arrays were designed for standard craniotomies used for cortical 2P-LSM in vivo imaging. Being of low weight, thin and flexible, they can be easily positioned between the dura mater and the glass coverslip. Three different designs were constructed: arrays (1) with eight circular electrodes (arranged in a matrix of three by three elements, sparing the center), (2) with sixteen circular electrodes (four by four matrix) and (3) with eight rectangular electrodes (placed in four groups of 2 single sites). The initial contact sites of gold were coated with nanoporous platinum to decrease the impedance of the electrode tissue contacts and to increase the charge transfer capability. The biocompatibility of the electrodes was confirmed by immuno-histochemistry. Electrical recordings and Ca2+-imaging were performed in mice with neuronal or astroglial expression of the genetically encoded Ca2+-sensor GCaMP3. With the sixteen channel electrode arrays, an estimation of the spatially resolved electrical activity pattern within the cranial window could be described. The eight channel arrays were used in studies for simultaneous acquisition of Ca2+ (using 2P-LSM) and electrical signals. In addition, Ca2+ signals could be elicited by electrical stimulation. Using different stimulation intensities and depth of anesthesia, the change of brain activity during transition from anesthetized to awake state was investigated. In addition, the LCP technology was transferred from the cortical to a spinal cord application.Neurone und Astrozyten bilden ein komplexes interagierendes zellulares Netzwerk zur Signalverarbeitung im Gehirn. Dabei sind die elektrische Aktivitäten der Nervenzellen und die Ca2+ Signale der Astrozyten eng aneinander gekoppelt. Parallele Aufzeichnungen der elektrischen Aktivität und der Ca2+ Signale können helfen, die molekularen Mechanismen der Neuron-Glia-Kommunikation zu identifizieren. Innerhalb dieser Arbeit wurden flexible Flüssigkristall-Polymer-Mikroelektrodenarrays für elektrische Aufzeichnungen und Stimulationen für die Zwei-Photonen-Laserscan- Mikroskopie (2P-LSM) entwickelt. Die Elektrodenarrays wurden für Standard-Kraniotomien entwickelt, die für die kortikale in vivo 2P-LSM verwendet werden. Sie sind dünn, flexibel und von geringem Gewicht und können leicht auf der Dura positioniert werden. Drei verschiedene Designs wurden konstruiert: Arrays (1) mit acht runden Elektroden (angeordnet in einer drei mal drei Matrix, ohne die mittlere Elektrode), (2) mit sechzehn kreisförmigen Elektroden (vier mal vier Matrix) und (3) mit acht rechteckigen Elektroden (angeordnet in vier Gruppen von zwei einzelnen Standorten). Die ursprünglichen Elektrodenkontakte aus Gold wurden mit nanoporösem Platin beschichtet, um die Gewebekontaktimpedanz zu verringern und die Ladungsübertragungsfähigkeit zu erhöhen. Die Biokompatibilität der Elektroden immunhistochemisch getestet. Elektrische Aktivität und Ca2+ Signale wurden bei Mäusen mit neuronaler oder astroglialer Expression des Ca2+-Indikators GCaMP3 aufgezeichnet. Mit den sechzehn Kanal-Elektroden-Arrays konnten die elektrische Aktivität entlang der Kortexoberfläche innerhalb der Kraniotomie charakterisiert werden. Die achtkanaligen Arrays wurden zur gleichzeitigen Erfassung von Ca2+ (mit 2P-LSM) und elektrischen Signalen verwendet. Darüber hinaus konnten Ca2+ Signale durch elektrische Stimulation hervorgerufen werden. Mit verschiedenen Stimulationsintensitäten und der Tiefe der Anästhesie (Isofluran) wurde die Veränderung der Hirnaktivität beim Übergang von anästhesiert zu wach beobachtet. Zusätzlich konnte die Flüssigkristall-Polymer -Technologie von der kortikalen auf die spinale Anwendung übertragen werden.- European Union / EUGlia-PhD - European Union / Neurofibre

Versatile Surface Electrodes for Combined Electrophysiology and Two-Photon Imaging of the Mouse Central Nervous System

Understanding and modulating CNS function in physiological as well as pathophysiological contexts remains a significant ambition in research and clinical applications. The investigation of the multifaceted CNS cell types including their interactions and contributions to neural function requires a combination of the state-ofthe-art in vivo electrophysiology and imaging techniques. We developed a novel type of liquid crystal polymer (LCP) surface micro-electrode manufactured in three customized designs with up to 16 channels for recording and stimulation of brain activity. All designs include spare central spaces for simultaneous 2P-imaging. Nanoporous platinumplated contact sites ensure a low impedance and high current transfer. The epidural implantation of the LCP micro-electrodes could be combined with standard cranial window surgery. The epidurally positioned electrodes did not only display long-term biocompatibility, but we also observed an additional stabilization of the underlying CNS tissue. We demonstrate the electrode’s versatility in combination with in vivo 2P-imaging by monitoring anesthesia-awake cycles of transgenic mice with GCaMP3 expression in neurons or astrocytes. Cortical stimulation and simultaneous 2P Ca2+ imaging in neurons or astrocytes highlighted the astrocytes’ integrative character in neuronal activity processing. Furthermore, we confirmed that spontaneous astroglial Ca2+ signals are dampened under anesthesia, while evoked signals in neurons and astrocytes showed stronger dependency on stimulation intensity rather than on various levels of anesthesia. Finally, we show that the electrodes provide recordings of the electrocorticogram (ECoG) with a high signal-to noise ratio and spatial signal differences which help to decipher brain activity states during experimental procedures. Summarizing, the novel LCP surface micro-electrode is a versatile, convenient, and reliable tool to investigate brain function in vivo

Comparative study of platinum electroplating to improve micro gold electrode arrays with LCP laminate

Decoding the cellular network interaction of neurons and glial cells are important in the development of new therapies for diseases of the central nervous system (CNS). Electrophysiological in vivo studies in mice will help to understand the highly complex network. In this paper, the optimization of epidural liquid crystal polymer (LCP) electrodes for different platinum electroplating parameters are presented and compared. Constant current and pulsed current electroplating varied in strength and duration was used to decrease the electrode impedance and to increase the charge storage capacity (CSCC). In best cases, both methods generated similar results with an impedance reduction of about 99%. However, electroplating with pulsed currents was less parameter-dependent than the electroplating with constant current. The use of ultrasound was essential to generate platinum coatings without plating defects. Electrode model parameters extracted from the electrode impedance reflected the increase in surface porosity due to the electroplating processes

Modeling and Simulations in Time Domain of a Stimulation Set-up for Cortical Applications

Electrical stimulation is used for example to treat neuronal disorders and depression with deep brain stimulation or transcranial electrical stimulation. Depending on the application, different electrodes are used and thus different electrical characteristics exist, which have to be handled by the stimulator. Without a measuring device the user would have to rely on the stimulator being able to deliver the needed stimulation signal. Therefore, the objective of this paper is to present a method to increase the level of confidence with characterization and modelling of the electrical behavior by using the example of one channel of our stimulation device for experimental use. In several simulation studies with an electrode model with values in a typical range for cortical applications the influence of the load onto the stimulator and the possibility to pre-estimate measuring signals in complex networks are shown

Improving electrocorticograms of awake and anaesthetized mice using wavelet denoising

The quality of bioelectrical signals is essential for functional evaluation of cellular circuits. The electrical activity recorded from the cortical brain surface represents the average of many individual synaptic processes. By downsizing micro-electrode arrays, the spatial resolution of electrocortico-grams (ECoGs) can be increased. But, upon increasing electrode impedance, recorded noise from the electrode-tissue interface and the surroundings will become more prominent. Frequently, signal interpretation is improved by post-processing using filtering or pattern recognition. For a variety of applications, wavelet denoising has become an accepted tool. Here, we present how wavelet denoising affects the signal-to-noise ratio of ECoGs. The recording qualities from awake and anesthetized mice was artificially reduced by adding two noise models prior to filtering. Raw and filtered signals were compared by calculating the linear correlation coefficient

Technical characterization of an 8 or 16 channel recording system to acquire electrocorticograms of mice

When performing electrocorticography, reliable recordings of bioelectrical signals are essential for signal processing and analysis. The acquisition of cellular electrical activity from the brain surface of mice requires a system that is able to record small signals within a low frequency range. This work presents a recording system with self-developed software and shows the result of a technical characterization in combination with self-developed electrode arrays to measure electrocorticograms of mice

Thymectomy in early childhood: significant alterations of the CD4+CD45RA+CD62L+ T cell compartment in later life (Figures)

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