Automated in vivo patch-clamp evaluation of extracellular multielectrode array spike recording capability.

Much innovation is currently aimed at improving the number, density, and geometry of electrodes on extracellular multielectrode arrays for in vivo recording of neural activity in the mammalian brain. To choose a multielectrode array configuration for a given neuroscience purpose, or to reveal design principles of future multielectrode arrays, it would be useful to have a systematic way of evaluating the spike recording capability of such arrays. We describe an automated system that performs robotic patch-clamp recording of a neuron being simultaneously recorded via an extracellular multielectrode array. By recording a patch-clamp data set from a neuron while acquiring extracellular recordings from the same neuron, we can evaluate how well the extracellular multielectrode array captures the spiking information from that neuron. To demonstrate the utility of our system, we show that it can provide data from the mammalian cortex to evaluate how the spike sorting performance of a close-packed extracellular multielectrode array is affected by bursting, which alters the shape and amplitude of spikes in a train. We also introduce an algorithmic framework to help evaluate how the number of electrodes in a multielectrode array affects spike sorting, examining how adding more electrodes yields data that can be spike sorted more easily. Our automated methodology may thus help with the evaluation of new electrode designs and configurations, providing empirical guidance on the kinds of electrodes that will be optimal for different brain regions, cell types, and species, for improving the accuracy of spike sorting. NEW & NOTEWORTHY We present an automated strategy for evaluating the spike recording performance of an extracellular multielectrode array, by enabling simultaneous recording of a neuron with both such an array and with patch clamp. We use our robot and accompanying algorithms to evaluate the performance of multielectrode arrays on supporting spike sorting.

Cell diversity and network dynamics in photosensitive human brain organoids.

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.

Close-Packed Silicon Microelectrodes for Scalable Spatially Oversampled Neural Recording.

OBJECTIVE: Neural recording electrodes are important tools for understanding neural codes and brain dynamics. Neural electrodes that are closely packed, such as in tetrodes, enable spatial oversampling of neural activity, which facilitates data analysis. Here we present the design and implementation of close-packed silicon microelectrodes to enable spatially oversampled recording of neural activity in a scalable fashion. METHODS: Our probes are fabricated in a hybrid lithography process, resulting in a dense array of recording sites connected to submicron dimension wiring. RESULTS: We demonstrate an implementation of a probe comprising 1000 electrode pads, each 9 × 9 µm, at a pitch of 11 µm. We introduce design automation and packaging methods that allow us to readily create a large variety of different designs. SIGNIFICANCE: We perform neural recordings with such probes in the live mammalian brain that illustrate the spatial oversampling potential of closely packed electrode sites.

Heterogeneous neural amplifier integration for scalable extracellular microelectrodes.

We here demonstrate multi-chip heterogeneous integration of microfabricated extracellular recording electrodes with neural amplifiers, highlighting a path to scaling electrode channel counts without the need for more complex monolithic integration. We characterize the noise and impedance performance of the heterogeneously integrated neural recording electrodes, and analyze the design parameters that enable the low-voltage neural input signals to co-exist with the high-frequency and high-voltage digital outputs on the same silicon substrate. This heterogeneous integration approach can enable future scaling efforts for microfabricated neural probes, and provides a design path for modular, fast, and independent scaling innovations in recording electrodes and neural amplifiers.

Computational reconstitution of spine calcium transients from individual proteins.

We have built a stochastic model in the program MCell that simulates Ca(2+) transients in spines from the principal molecular components believed to control Ca(2+) entry and exit. Proteins, with their kinetic models, are located within two segments of dendrites containing 88 intact spines, centered in a fully reconstructed 6 × 6 × 5 µm(3) cube of hippocampal neuropil. Protein components include AMPA- and NMDA-type glutamate receptors, L- and R-type voltage-dependent Ca(2+) channels, Na(+)/Ca(2+) exchangers, plasma membrane Ca(2+) ATPases, smooth endoplasmic reticulum Ca(2+) ATPases, immobile Ca(2+) buffers, and calbindin. Kinetic models for each protein were taken from published studies of the isolated proteins in vitro. For simulation of electrical stimuli, the time course of voltage changes in the dendritic spine was generated with the desired stimulus in the program NEURON. Voltage-dependent parameters were then continuously re-adjusted during simulations in MCell to reproduce the effects of the stimulus. Nine parameters of the model were optimized within realistic experimental limits by a process that compared results of simulations to published data. We find that simulations in the optimized model reproduce the timing and amplitude of Ca(2+) transients measured experimentally in intact neurons. Thus, we demonstrate that the characteristics of individual isolated proteins determined in vitro can accurately reproduce the dynamics of experimentally measured Ca(2+) transients in spines. The model will provide a test bed for exploring the roles of additional proteins that regulate Ca(2+) influx into spines and for studying the behavior of protein targets in the spine that are regulated by Ca(2+) influx.

A direct-to-drive neural data acquisition system.

Driven by the increasing channel count of neural probes, there is much effort being directed to creating increasingly scalable electrophysiology data acquisition (DAQ) systems. However, all such systems still rely on personal computers for data storage, and thus are limited by the bandwidth and cost of the computers, especially as the scale of recording increases. Here we present a novel architecture in which a digital processor receives data from an analog-to-digital converter, and writes that data directly to hard drives, without the need for a personal computer to serve as an intermediary in the DAQ process. This minimalist architecture may support exceptionally high data throughput, without incurring costs to support unnecessary hardware and overhead associated with personal computers, thus facilitating scaling of electrophysiological recording in the future.

Closed-loop, ultraprecise, automated craniotomies.

A large array of neuroscientific techniques, including in vivo electrophysiology, two-photon imaging, optogenetics, lesions, and microdialysis, require access to the brain through the skull. Ideally, the necessary craniotomies could be performed in a repeatable and automated fashion, without damaging the underlying brain tissue. Here we report that when drilling through the skull a stereotypical increase in conductance can be observed when the drill bit passes through the skull base. We present an architecture for a robotic device that can perform this algorithm, along with two implementations--one based on homebuilt hardware and one based on commercially available hardware--that can automatically detect such changes and create large numbers of precise craniotomies, even in a single skull. We also show that this technique can be adapted to automatically drill cranial windows several millimeters in diameter. Such robots will not only be useful for helping neuroscientists perform both small and large craniotomies more reliably but can also be used to create precisely aligned arrays of craniotomies with stereotaxic registration to standard brain atlases that would be difficult to drill by hand.

Extracellular sheets and tunnels modulate glutamate diffusion in hippocampal neuropil.

Although the extracellular space in the neuropil of the brain is an important channel for volume communication between cells and has other important functions, its morphology on the micron scale has not been analyzed quantitatively owing to experimental limitations. We used manual and computational techniques to reconstruct the 3D geometry of 180 µm(3) of rat CA1 hippocampal neuropil from serial electron microscopy and corrected for tissue shrinkage to reflect the in vivo state. The reconstruction revealed an interconnected network of 40-80 nm diameter tunnels, formed at the junction of three or more cellular processes, spanned by sheets between pairs of cell surfaces with 10-40 nm width. The tunnels tended to occur around synapses and axons, and the sheets were enriched around astrocytes. Monte Carlo simulations of diffusion within the reconstructed neuropil demonstrate that the rate of diffusion of neurotransmitter and other small molecules was slower in sheets than in tunnels. Thus, the non-uniformity found in the extracellular space may have specialized functions for signaling (sheets) and volume transmission (tunnels).