Technologies to Study Action Potential Propagation With a Focus on HD-MEAs

Axons convey information in neuronal circuits via reliable conduction of action potentials (APs) from the axon initial segment (AIS) to the presynaptic terminals. Recent experimental findings increasingly evidence that the axonal function is not limited to the simple transmission of APs. Advances in subcellular-resolution recording techniques have shown that axons display activity-dependent modulation in spike shape and conduction velocity, which influence synaptic strength and latency. We briefly review here, how recent methodological developments facilitate the understanding of the axon physiology. We included the three most common methods, i.e., genetically encoded voltage imaging (GEVI), subcellular patch-clamp and high-density microelectrode arrays (HD-MEAs). We then describe the potential of using HD-MEAs in studying axonal physiology in more detail. Due to their robustness, amenability to high-throughput and high spatiotemporal resolution, HD-MEAs can provide a direct functional electrical readout of single cells and cellular ensembles at subcellular resolution. HD-MEAs can, therefore, be employed in investigating axonal pathologies, the effects of large-scale genomic interventions (e.g., with RNAi or CRISPR) or in compound screenings. A combination of extracellular microelectrode arrays (MEAs), intracellular microelectrodes and optical imaging may potentially reveal yet unexplored repertoires of axonal functions

Revealing the distribution of transmembrane currents along the dendritic tree of a neuron from extracellular recordings.

Revealing the current source distribution along the neuronal membrane is a key step on the way to understanding neural computations, however, the experimental and theoretical tools to achieve sufficient spatiotemporal resolution for the estimation remain to be established. Here we address this problem using extracellularly recorded potentials with arbitrarily distributed electrodes for a neuron of known morphology. We use simulations of models with varying complexity to validate the proposed method and to give recommendations for experimental applications. The method is applied to in vitro data from rat hippocampus

Materials and neuroscience: validating tools for large-scale, high-density neural recording

Extracellular recording remains the only technique capable of measuring the activity of many neurons simultaneously with a sub-millisecond precision, in multiple brain areas, including deep structures. Nevertheless, many questions about the nature of the detected signal and the limitations/capabilities of this technique remain unanswered. The general goal of this work is to apply the methodology and concepts of materials science to answer some of the major questions surrounding extracellular recording, and thus take full advantage of this seminal technique. We start out by quantifying the effect of electrode impedance on the amplitude of measured extracellular spikes and background noise. Can we improve data quality by lowering electrode impedance? We demonstrate that if the proper recording system is used, then the impedance of a microelectrode, within the range typical of standard polytrodes (~ 0.1 to 2 MΩ), does not significantly affect a neural spike amplitude or the background noise, and therefore spike sorting. In addition to improving the performance of each electrode, increasing the number of electrodes in a single neural probe has also proven advantageous for simultaneously monitoring the activity of more neurons with better spatiotemporal resolution. How can we achieve large-scale, highdensity extracellular recordings without compromising brain tissue? Here we report the design and in vivo validation of a complementary metal–oxide–semiconductor (CMOS)-based scanning probe with 1356 electrodes arranged along approximately 8 mm of a thin shaft (50 μm thick and 100 μm wide). Additionally, given the ever-shrinking dimensions of CMOS technology, there is a drive to fabricate sub-cellular electrodes (< 10 μm). Therefore, to evaluate electrode configurations for future probe designs, several recordings from many different brain regions were performed with an ultra-dense probe containing 255 electrodes, each with a geometric area of 5 x 5 μm and a pitch of 6 μm. How can we validate neural probes with different electrode materials/configurations and different sorting algorithms? We describe a new procedure for precisely aligning two probes for in vivo “paired-recordings” such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micro-pipette. We gathered a dataset of paired-recordings, which is available online. The “ground truth” data, for which one knows exactly when a neuron in the vicinity of an extracellular probe generates an action potential, has been used for several groups to validate and quantify the performance of new algorithms to automatically detect/sort single-units

Organic electrochemical transistors based on PEDOT:PSS for the sensing of cellular signals from confluent cell layers down to single cells

Einleitung: Organisch elektrochemische Transistoren basierend auf dem Polymer poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) sind Biosensoren, welche p-Typ Transistor Charakteristika zeigen basierend aus der Bewegung von Kationen in und aus der Polymerschicht. Die Sensorkonfiguration besteht aus drei Kontakten: der Source, dem Drain und der Gate-Elektrode, wobei die Polymerschicht durch einen Elektrolyten von der Gate-Elektrode getrennt ist. Die Kationen aus dem Elektrolyten werden durch die angelegte Spannung in das PEDOT:PSS geleitet, wo sie die offenen Sulfonat-Anionen des PSS kompensieren. Dies wiederum erhöht die Dichte der Löcher im PEDOT, was zu einem Abfall des Drain-Stroms führt. Dieser Stromabfall resultiert in der Ausschaltung des Sensors. Dieses Sensorverhalten kann für die unterschiedlichsten biologischen Messungen verwendet werden. Die OECTs können für die Detektion von elektrisch aktiven Zellen genutzt werden und erlauben gleichzeitig auch die Messung der Zelladhäsion. Die Nutzung dieser Sensoren für die Messung von Daten aus konfluenten Zellschichten bis zu Einzelzellmessungen in Kombination mit einer mathematischen Beschreibung der Ergebnisse wurde bisher noch nicht gezeigt. Ergebnisse: Um universal einsetzbare, hoch-sensitive und transparente Sensoren zu produzieren, wurden etablierte Reinraumprozesse in neuer und vereinfachter Weise genutzt. Faktoren die während der Sensorherstellung zu Schädigungen der Polymerschicht führen könnten, wie z.B. Ultraviolettstrahlung, wurden komplett eliminiert. Die Sensoren wurden bezüglich ihrer elektrischen Fähigkeiten und ihrer Stabilität in nassen sowie trockenen Umständen getestet. Das Testverfahren ermöglichte die Festsetzung der optimalen Parameter für die Herstellung der organisch elektrochemische Transistoren. Als wichtigster Faktor für das Sensorverhalten wurde das Volumen der Polymerschicht bestimmt. Das Volumen des PEDOT:PSS bestimmt die elektrischen Eigenschaften der Sensoren. Bleibt das Volumen der Polymerschicht für die Sensoren konstant, so wird die gleiche Transkonduktanz gemessen, eine Änderung in der Schichtdicke führt jedoch zu einem andern Verhalten bezüglich der Grenzfrequenz. Dünnere Schichten zeigen eine Erhöhung der Grenzfrequenz, wobei dickere Schichten einen gegenteiligen Effekt zeigen. Aus diesem Grund musste ein optimiertes Design erstellt werden, um die richtige Funktion der Sensoren für die geplanten Experimente zu gewährleisten. Unterschiedliche Zelltypen wurden genutzt, um ein breites Spektrum an Anwendungen für die fabrizierten Sensoren zu testen. Herzzellen wurden für die Messung von extrazellulären Aktionspotenzialen eingesetzt. Die getesteten Sensoren zeigten ein sehr gutes Signal-Rausch-Verhältnis mit schnellen Messzeiten, was sie zu idealen Sensoren für Aktionspotenzialmessungen macht. Zur selben Zeit wurden Transistor-Transferfunktionsmessungen durchgeführt, um die Fähigkeiten der Sensoren im Bereich der Impedanzmessungen zu ergründen. Diese Art von Messungen wurde bisher noch nicht publiziert. Durch die Verwendung von dicht wachsenden Madin-Darbey Kidney Zellen konnte die Änderung der Zellimpedanz durch Änderungen in den Zellverbindungen gemessen werden. Im Gegensatz zu den Madin-Darbey Kidney Zellen wachsen Human Embryo Kidney Zellen ohne Zellverbindungen. Da die Messung von dichten Zellkulturen nur Aussagen über die Population von Zellen als Ganzes erlaubt, wurden neue Protokolle entwickelt, um auf Einzelzelllevel zu messen. Die organisch elektrochemische Transistoren zeigten die Fähigkeit, Aktionspotenziale von Zellen sowie deren Adhäsion mit hoher Reproduzierbarkeit und Präzision zu messen. Organisch elektrochemische Transistoren, die die Transistor-Transferfunktion bis hinunter auf Einzelzellebene nutzen wurden bisher noch nicht gezeigt. Zusätzlich wurde ein mathematisches Modell entwickelt, um die Zellparameter aus den gewonnenen Daten zu ermitteln. Das mathematische Modell dient dabei der Verbesserung des Verständnisses bezüglich der Interaktion von Zellen und den Sensoren. Die Kombination aus den gezeigten Biosensoren mit optischer Transparenz und der Möglichkeit des mathematischen Fittens der Daten erlauben die Möglichkeit für unzählige Experimente. Ausblick: Die gezeigten Sensoren bieten eine exzellente Plattform für die Biosensorik mit der Möglichkeit für viele zukünftige Anwendungen. Die Sensoren sind dabei nicht auf die gezeigten Anwendungen limitiert, sondern können mit einfachen Mitteln für die unterschiedlichsten Zwecke angepasst werden.Summary: Organic electrochemical transistors based on the polymer poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) are biosensors which use the movement of cations into and out of the polymer layer to generate a behavior that mimics p-type transistors. The device configuration has a source contact, a drain contact, and a gate electrode, which is separated from the polymer layer by an electrolyte. The cations of the electrolyte enter the PEDOT:PSS and compensate the pendant sulfonate anions on the PSS which increases the hole density in PEDOT. This results in a decrease of the drain current and a switching of the device into the off state. Using this device behavior, several biological signals can be detected. The OECTs can be used for the detection of action potentials of electrogenic cells, but also enable the measurement of the adhesion of cells to the device. The utilization of these devices for the measurement of confluent cell layers down to single cells in combination with a mathematical description was not shown so far. Results: In order to achieve versatile, highly sensitive, and transparent sensors, the fabrication of the devices with standard cleanroom processes was established in a unique and simplified way. Deteriorating factors such as exposure to ultraviolet radiation and contact with water were eliminated from the fabrication process. The sensors were characterized in regards to their electrical performance and stability in dry and wet conditions. The gathered results were used to generate a protocol for the best performing chips. Based on the generated data protocols for the fabrication, chemical post-treatment, as well as device operation, were established. Different sensing areas of the polymer layer were tested to determine their advantages for biosensing. The crucial factor for the devices was based on the volume of the deposited polymer layer. By keeping the volume of the PEDOT:PSS constant the transconductance remains the same, however thicker PEDOT:PSS layers resulted in devices with a comparatively lower cutoff frequency while thinner polymer layers resulted in a comparatively higher cutoff frequency. Therefore, an optimized chip layout had to be made to guarantee the functionality of the devices for their applications. Different cell types were used to test the devices towards their cell-sensing capabilities. Cardiomyocytes were used to establish the sensors for action potential measurements, and it was found that the sensors inherit a high signal-to-noise ratio making these devices ideal candidates for action potential measurements. At the same time, the impedimetric capabilities of the devices were investigated according to transistor-transfer function measurements which were not shown before with PEDOT:PSS based organic electrochemical transistors. By using densely growing cells, such as the Madin-Darby canine kidney cells, the change in impedance spectra towards changes in gap junction resistance could be proven. Human embryo kidney cells were used to investigate the behavior of dense cell cultures when no gap junctions are present. Since the observation of dense cellular cultures only allows for experiments on an arbitrary amount of cells, a protocol was established, and the devices were tested for measurements on a single cell level. The devices showed the capability for measurements of action potentials with the additional impedimetric data in high precision and reproducibility. Devices utilizing transistor-transfer function measurements with organic electrochemical transistors down to single cell level have not been shown so far. In addition, a new mathematical model was developed in order to calculate the cell-related parameters which demonstrate the distance between the cell and the polymer, offering a closer insight into the cellular attachment and detachment behavior. In combination with the fitting, the present platform was established with several possible applications ranging from confluent cells down to single cells while also offering the possibility of optically controlling the cell behavior due to the transparency of the devices. Outlook: The established devices offer an excellent biosensing platform which can be used in several future applications. The devices are not limited to the shown applications and can be altered to fit the desired use

Bioelectronic Medicine: a multidisciplinary roadmap from biophysics to precision therapies

Bioelectronic Medicine stands as an emerging field that rapidly evolves and offers distinctive clinical benefits, alongside unique challenges. It consists of the modulation of the nervous system by precise delivery of electrical current for the treatment of clinical conditions, such as post-stroke movement recovery or drug-resistant disorders. The unquestionable clinical impact of Bioelectronic Medicine is underscored by the successful translation to humans in the last decades, and the long list of preclinical studies. Given the emergency of accelerating the progress in new neuromodulation treatments (i.e., drug-resistant hypertension, autoimmune and degenerative diseases), collaboration between multiple fields is imperative. This work intends to foster multidisciplinary work and bring together different fields to provide the fundamental basis underlying Bioelectronic Medicine. In this review we will go from the biophysics of the cell membrane, which we consider the inner core of neuromodulation, to patient care. We will discuss the recently discovered mechanism of neurotransmission switching and how it will impact neuromodulation design, and we will provide an update on neuronal and glial basis in health and disease. The advances in biomedical technology have facilitated the collection of large amounts of data, thereby introducing new challenges in data analysis. We will discuss the current approaches and challenges in high throughput data analysis, encompassing big data, networks, artificial intelligence, and internet of things. Emphasis will be placed on understanding the electrochemical properties of neural interfaces, along with the integration of biocompatible and reliable materials and compliance with biomedical regulations for translational applications. Preclinical validation is foundational to the translational process, and we will discuss the critical aspects of such animal studies. Finally, we will focus on the patient point-of-care and challenges in neuromodulation as the ultimate goal of bioelectronic medicine. This review is a call to scientists from different fields to work together with a common endeavor: accelerate the decoding and modulation of the nervous system in a new era of therapeutic possibilities

Respiratory Control: Central and Peripheral Mechanisms

Understanding of the respiratory control system has been greatly improved by technological and methodological advances. This volume integrates results from many perspectives, brings together diverse approaches to the investigations, and represents important additions to the field of neural control of breathing. Topics include membrane properties of respiratory neurons, in vitro studies of respiratory control, chemical neuroanatomy, central integration of respiratory afferents, modulation of respiratory pattern by peripheral afferents, respiratory chemoreception, development of respiratory control, behavioral control of breathing, and human ventilatory control. Forty-seven experts in the field report research and discuss novel issues facing future investigations in this collection of papers from an international conference of nearly two hundred leading scientists held in October 1990. This research is of vital importance to respiratory physiologists and those in neurosciences and neurobiology who work with integrative sensory and motor systems and is pertinent to both basic and clinical investigations. Respiratory Control is destined to be widely cited because of the strength of the contributors and the dearth of similar works. The four editors are affiliated with the University of Kentucky: Dexter F. Speck is associate professor of physiology and biophysics, Michael S. Dekin is assistant professor of biological sciences, W. Robert Revelette is research scientist of physiology and biophysics, and Donald T. Frazier is professor and chairman of physiology and biophysics. Experts in the field report current research and discuss novel issues facing future investigations. —SciTech Book Newshttps://uknowledge.uky.edu/upk_biology/1002/thumbnail.jp

NEURAL CIRCUIT DYNAMICS AND FUNCTION OF COMPLEX BEHAVIORAL STATES

Mammalian neural circuits are sophisticated biological systems that choreograph behavioral processes vital for survival. While the inherent complexity of discrete neural circuits has proven difficult to decipher, many parallel methodological developments promise to help delineate the function and connectivity of molecularly defined neural circuits. Here, I utilize novel neurotechniques to precisely monitor and manipulate anxiety- and feeding-related circuit activity. By using a holistic, multifaceted approach for perturbing and measuring neural circuit dynamics, we begin to provide a framework for understanding how adaptive and maladaptive behavioral states are manifested through the cooperative interactions of discrete extended amygdala, midbrain, and hypothalamic circuit elements.Doctor of Philosoph