A Multi-Functional Microelectrode Array Featuring 59760 Electrodes, 2048 Electrophysiology Channels, Stimulation, Impedance Measurement and Neurotransmitter Detection Channels.

Biological cells are characterized by highly complex phenomena and processes that are, to a great extent, interdependent. To gain detailed insights, devices designed to study cellular phenomena need to enable tracking and manipulation of multiple cell parameters in parallel; they have to provide high signal quality and high spatiotemporal resolution. To this end, we have developed a CMOS-based microelectrode array system that integrates six measurement and stimulation functions, the largest number to date. Moreover, the system features the largest active electrode array area to date (4.48×2.43 mm2) to accommodate 59,760 electrodes, while its power consumption, noise characteristics, and spatial resolution (13.5 µm electrode pitch) are comparable to the best state-of-the-art devices. The system includes: 2,048 action-potential (AP, bandwidth: 300 Hz to 10 kHz) recording units, 32 local-field-potential (LFP, bandwidth: 1 Hz to 300 Hz) recording units, 32 current recording units, 32 impedance measurement units, and 28 neurotransmitter detection units, in addition to the 16 dual-mode voltage-only or current/voltage-controlled stimulation units. The electrode array architecture is based on a switch matrix, which allows for connecting any measurement/stimulation unit to any electrode in the array and for performing different measurement/stimulation functions in parallel.

A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro.

To advance our understanding of the functioning of neuronal ensembles, systems are needed to enable simultaneous recording from a large number of individual neurons at high spatiotemporal resolution and good signal-to-noise ratio. Moreover, stimulation capability is highly desirable for investigating, for example, plasticity and learning processes. Here, we present a microelectrode array (MEA) system on a single CMOS die for in vitro recording and stimulation. The system incorporates 26,400 platinum electrodes, fabricated by in-house post-processing, over a large sensing area (3.85 × 2.10 mm2) with sub-cellular spatial resolution (pitch of 17.5 µm). Owing to an area and power efficient implementation, we were able to integrate 1024 readout channels on chip to record extracellular signals from a user-specified selection of electrodes. These channels feature noise values of 2.4 µVrms in the action-potential band (300 Hz-10 kHz) and 5.4 µVrms in the local-field-potential band (1 Hz-300 Hz), and provide programmable gain (up to 78 dB) to accommodate various biological preparations. Amplified and filtered signals are digitized by 10 bit parallel single-slope ADCs at 20 kSamples/s. The system also includes 32 stimulation units, which can elicit neural spikes through either current or voltage pulses. The chip consumes only 75 mW in total, which obviates the need of active cooling even for sensitive cell cultures.

Functional imaging of conduction dynamics in cortical and spinal axons.

Mammalian axons are specialized for transmitting action potentials to targets within the central and peripheral nervous system. A growing body of evidence suggests that, besides signal conduction, axons play essential roles in neural information processing, and their malfunctions are common hallmarks of neurodegenerative diseases. The technologies available to study axonal function and structure integrally limit the comprehension of axon neurobiology. High-density microelectrode arrays (HD-MEAs) allow for accessing axonal action potentials at high spatiotemporal resolution, but provide no insights on axonal morphology. Here, we demonstrate a method for electrical visualization of axonal morphologies based on extracellular action potentials recorded from cortical and motor neurons using HD-MEAs. The method enabled us to reconstruct up to 5-cm-long axonal arbors and directly monitor axonal conduction across thousands of recording sites. We reconstructed 1.86 m of cortical and spinal axons in total and found specific features in their structure and function.

Large-Scale Mapping of Axonal Arbors Using High-Density Microelectrode Arrays.

Understanding the role of axons in neuronal information processing is a fundamental task in neuroscience. Over the last years, sophisticated patch-clamp investigations have provided unexpected and exciting data on axonal phenomena and functioning, but there is still a need for methods to investigate full axonal arbors at sufficient throughput. Here, we present a new method for the simultaneous mapping of the axonal arbors of a large number of individual neurons, which relies on their extracellular signals that have been recorded with high-density microelectrode arrays (HD-MEAs). The segmentation of axons was performed based on the local correlation of extracellular signals. Comparison of the results with both, ground truth and receiver operator characteristics, shows that the new segmentation method outperforms previously used methods. Using a standard HD-MEA, we mapped the axonal arbors of 68 neurons in <6 h. The fully automated method can be extended to new generations of HD-MEAs with larger data output and is estimated to provide data of axonal arbors of thousands of neurons within recording sessions of a few hours.

The Axon Initial Segment is the Dominant Contributor to the Neuron's Extracellular Electrical Potential Landscape.

Extracellular voltage fields, produced by a neuron's action potentials, provide a widely used means for studying neuronal and neuronal-network function. The neuron's soma and dendrites are thought to drive the extracellular action potential (EAP) landscape, while the axon's contribution is usually considered less important. However, by recording voltages of single neurons in dissociated rat cortical cultures and Purkinje cells in acute mouse cerebellar slices through hundreds of densely packed electrodes, it is found, instead, that the axon initial segment dominates the measured EAP landscape, and, surprisingly, the soma only contributes to a minor extent. As expected, the recorded dominant signal has negative polarity (charge entering the cell) and initiates at the distal end. Interestingly, signals with positive polarity (charge exiting the cell) occur near some but not all dendritic branches and occur after a delay. Such basic knowledge about which neuronal compartments contribute to the extracellular voltage landscape is important for interpreting results from all electrical readout schemes. Finally, initiation of the electrical activity at the distal end of the axon initial segment (AIS) and subsequent spreading into the axon proper and backward through the proximal AIS toward the soma are confirmed. The corresponding extracellular waveforms across different neuronal compartments could be tracked.

Stimulation and Artifact-Suppression Techniques for In Vitro High-Density Microelectrode Array Systems.

We present novel voltage stimulation buffers with controlled output current, along with recording circuits featuring adjustable high-pass cut-off filtering to perform efficient stimulation while actively suppressing stimulation artifacts in high-density microelectrode arrays. Owing to the dense packing and close proximity of the electrodes in such systems, a stimulation through one electrode can cause large electrical artifacts on neighboring electrodes that easily saturate the corresponding recording amplifiers. To suppress such artifacts, the high-pass corner frequencies of all available 2048 recording channels can be raised from several Hz to several kHz by applying a "soft-reset" or pole-shifting technique. With the implemented artifact suppression technique, the saturation time of the recording circuits, connected to electrodes in immediate vicinity to the stimulation site, could be reduced to less than 150 µs. For the stimulation buffer, we developed a circuit, which can operate in two modes: either control of only the stimulation voltage or control of current and voltage during stimulation. The voltage-only controlled mode employs a local common-mode feedback operational transconductance amplifier with a near rail-to-rail input/output range, suitable for driving high-capacitive loads. The current/voltage controlled mode is based on a positive current conveyor generating adjustable output currents, whereas its upper and lower output voltages are limited by two feedback loops. The current/voltage controlled circuit can generate stimulation pulses up to 30 µA with less than ±0.1% linearity error in the low-current mode and up to 300 µA with less than ±0.2% linearity error in the high-current mode.

Combination of High-density Microelectrode Array and Patch Clamp Recordings to Enable Studies of Multisynaptic Integration.

We present a novel, all-electric approach to record and to precisely control the activity of tens of individual presynaptic neurons. The method allows for parallel mapping of the efficacy of multiple synapses and of the resulting dynamics of postsynaptic neurons in a cortical culture. For the measurements, we combine an extracellular high-density microelectrode array, featuring 11'000 electrodes for extracellular recording and stimulation, with intracellular patch-clamp recording. We are able to identify the contributions of individual presynaptic neurons - including inhibitory and excitatory synaptic inputs - to postsynaptic potentials, which enables us to study dendritic integration. Since the electrical stimuli can be controlled at microsecond resolution, our method enables to evoke action potentials at tens of presynaptic cells in precisely orchestrated sequences of high reliability and minimum jitter. We demonstrate the potential of this method by evoking short- and long-term synaptic plasticity through manipulation of multiple synaptic inputs to a specific neuron.

Tracking individual action potentials throughout mammalian axonal arbors.

Axons are neuronal processes specialized for conduction of action potentials (APs). The timing and temporal precision of APs when they reach each of the synapses are fundamentally important for information processing in the brain. Due to small diameters of axons, direct recording of single AP transmission is challenging. Consequently, most knowledge about axonal conductance derives from modeling studies or indirect measurements. We demonstrate a method to noninvasively and directly record individual APs propagating along millimeter-length axonal arbors in cortical cultures with hundreds of microelectrodes at microsecond temporal resolution. We find that cortical axons conduct single APs with high temporal precision (~100 µs arrival time jitter per mm length) and reliability: in more than 8,000,000 recorded APs, we did not observe any conduction or branch-point failures. Upon high-frequency stimulation at 100 Hz, successive became slower, and their arrival time precision decreased by 20% and 12% for the 100th AP, respectively.

Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation.

Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10-20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations.

Electrical Identification and Selective Microstimulation of Neuronal Compartments Based on Features of Extracellular Action Potentials.

A detailed, high-spatiotemporal-resolution characterization of neuronal responses to local electrical fields and the capability of precise extracellular microstimulation of selected neurons are pivotal for studying and manipulating neuronal activity and circuits in networks and for developing neural prosthetics. Here, we studied cultured neocortical neurons by using high-density microelectrode arrays and optical imaging, complemented by the patch-clamp technique, and with the aim to correlate morphological and electrical features of neuronal compartments with their responsiveness to extracellular stimulation. We developed strategies to electrically identify any neuron in the network, while subcellular spatial resolution recording of extracellular action potential (AP) traces enabled their assignment to the axon initial segment (AIS), axonal arbor and proximal somatodendritic compartments. Stimulation at the AIS required low voltages and provided immediate, selective and reliable neuronal activation, whereas stimulation at the soma required high voltages and produced delayed and unreliable responses. Subthreshold stimulation at the soma depolarized the somatic membrane potential without eliciting APs.

Multiple Single-Unit Long-Term Tracking on Organotypic Hippocampal Slices Using High-Density Microelectrode Arrays.

A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed "footprints" of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times.

High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels.

Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 µm) within a large overall sensing area (3.85 × 2.10 mm(2)). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons.

Parameters for burst detection.

Bursts of action potentials within neurons and throughout networks are believed to serve roles in how neurons handle and store information, both in vivo and in vitro. Accurate detection of burst occurrences and durations are therefore crucial for many studies. A number of algorithms have been proposed to do so, but a standard method has not been adopted. This is due, in part, to many algorithms requiring the adjustment of multiple ad-hoc parameters and further post-hoc criteria in order to produce satisfactory results. Here, we broadly catalog existing approaches and present a new approach requiring the selection of only a single parameter: the number of spikes N comprising the smallest burst to consider. A burst was identified if N spikes occurred in less than T ms, where the threshold T was automatically determined from observing a probability distribution of inter-spike-intervals. Performance was compared vs. different classes of detectors on data gathered from in vitro neuronal networks grown over microelectrode arrays. Our approach offered a number of useful features including: a simple implementation, no need for ad-hoc or post-hoc criteria, and precise assignment of burst boundary time points. Unlike existing approaches, detection was not biased toward larger bursts, allowing identification and analysis of a greater range of neuronal and network dynamics.

Tracking axonal action potential propagation on a high-density microelectrode array across hundreds of sites.

Axons are traditionally considered stable transmission cables, but evidence of the regulation of action potential propagation demonstrates that axons may have more important roles. However, their small diameters render intracellular recordings challenging, and low-magnitude extracellular signals are difficult to detect and assign. Better experimental access to axonal function would help to advance this field. Here we report methods to electrically visualize action potential propagation and network topology in cortical neurons grown over custom arrays, which contain 11,011 microelectrodes and are fabricated using complementary metal oxide semiconductor technology. Any neuron lying on the array can be recorded at high spatio-temporal resolution, and simultaneously precisely stimulated with little artifact. We find substantial velocity differences occurring locally within single axons, suggesting that the temporal control of a neuron's output may contribute to neuronal information processing.

High-density microelectrode array recordings and real-time spike sorting for closed-loop experiments: an emerging technology to study neural plasticity.

Understanding plasticity of neural networks is a key to comprehending their development and function. A powerful technique to study neural plasticity includes recording and control of pre- and post-synaptic neural activity, e.g., by using simultaneous intracellular recording and stimulation of several neurons. Intracellular recording is, however, a demanding technique and has its limitations in that only a small number of neurons can be stimulated and recorded from at the same time. Extracellular techniques offer the possibility to simultaneously record from larger numbers of neurons with relative ease, at the expenses of increased efforts to sort out single neuronal activities from the recorded mixture, which is a time consuming and error prone step, referred to as spike sorting. In this mini-review, we describe recent technological developments in two separate fields, namely CMOS-based high-density microelectrode arrays, which also allow for extracellular stimulation of neurons, and real-time spike sorting. We argue that these techniques, when combined, will provide a powerful tool to study plasticity in neural networks consisting of several thousand neurons in vitro.