Recurrently connected and localized neuronal communities initiate coordinated spontaneous activity in neuronal networks.

Developing neuronal systems intrinsically generate coordinated spontaneous activity that propagates by involving a large number of synchronously firing neurons. In vivo, waves of spikes transiently characterize the activity of developing brain circuits and are fundamental for activity-dependent circuit formation. In vitro, coordinated spontaneous spiking activity, or network bursts (NBs), interleaved within periods of asynchronous spikes emerge during the development of 2D and 3D neuronal cultures. Several studies have investigated this type of activity and its dynamics, but how a neuronal system generates these coordinated events remains unclear. Here, we investigate at a cellular level the generation of network bursts in spontaneously active neuronal cultures by exploiting high-resolution multielectrode array recordings and computational network modelling. Our analysis reveals that NBs are generated in specialized regions of the network (functional neuronal communities) that feature neuronal links with high cross-correlation peak values, sub-millisecond lags and that share very similar structural connectivity motifs providing recurrent interactions. We show that the particular properties of these local structures enable locally amplifying spontaneous asynchronous spikes and that this mechanism can lead to the initiation of NBs. Through the analysis of simulated and experimental data, we also show that AMPA currents drive the coordinated activity, while NMDA and GABA currents are only involved in shaping the dynamics of NBs. Overall, our results suggest that the presence of functional neuronal communities with recurrent local connections allows a neuronal system to generate spontaneous coordinated spiking activity events. As suggested by the rules used for implementing our computational model, such functional communities might naturally emerge during network development by following simple constraints on distance-based connectivity.

Intracellular and Extracellular Recording of Spontaneous Action Potentials in Mammalian Neurons and Cardiac Cells with 3D Plasmonic Nanoelectrodes.

Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.

Sloppiness in spontaneously active neuronal networks.

Various plasticity mechanisms, including experience-dependent, spontaneous, as well as homeostatic ones, continuously remodel neural circuits. Yet, despite fluctuations in the properties of single neurons and synapses, the behavior and function of neuronal assemblies are generally found to be very stable over time. This raises the important question of how plasticity is coordinated across the network. To address this, we investigated the stability of network activity in cultured rat hippocampal neurons recorded with high-density multielectrode arrays over several days. We used parametric models to characterize multineuron activity patterns and analyzed their sensitivity to changes. We found that the models exhibited sloppiness, a property where the model behavior is insensitive to changes in many parameter combinations, but very sensitive to a few. The activity of neurons with sloppy parameters showed faster and larger fluctuations than the activity of a small subset of neurons associated with sensitive parameters. Furthermore, parameter sensitivity was highly correlated with firing rates. Finally, we tested our observations from cell cultures on an in vivo recording from monkey visual cortex and we confirm that spontaneous cortical activity also shows hallmarks of sloppy behavior and firing rate dependence. Our findings suggest that a small subnetwork of highly active and stable neurons supports group stability, and that this endows neuronal networks with the flexibility to continuously remodel without compromising stability and function.

Specific Neuron Placement on Gold and Silicon Nitride-Patterned Substrates through a Two-Step Functionalization Method.

The control of neuron-substrate adhesion has been always a challenge for fabricating neuron-based cell chips and in particular for multielectrode array (MEA) devices, which warrants the investigation of the electrophysiological activity of neuronal networks. The recent introduction of high-density chips based on the complementary metal oxide semiconductor (CMOS) technology, integrating thousands of electrodes, improved the possibility to sense large networks and raised the challenge to develop newly adapted functionalization techniques to further increase neuron electrode localization to avoid the positioning of cells out of the recording area. Here, we present a simple and straightforward chemical functionalization method that leads to the precise and exclusive positioning of the neural cell bodies onto modified electrodes and inhibits, at the same time, cellular adhesion in the surrounding insulator areas. Different from other approaches, this technique does not require any adhesion molecule as well as complex patterning technique such as µ-contact printing. The functionalization was first optimized on gold (Au) and silicon nitride (Si3N4)-patterned surfaces. The procedure consisted of the introduction of a passivating layer of hydrophobic silane molecules (propyltriethoxysilane [PTES]) followed by a treatment of the Au surface using 11-amino-1-undecanethiol hydrochloride (AT). On model substrates, well-ordered neural networks and an optimal coupling between a single neuron and single micrometric functionalized Au surface were achieved. In addition, we presented the preliminary results of this functionalization method directly applied on a CMOS-MEA: the electrical spontaneous spiking and bursting activities of the network recorded for up to 4 weeks demonstrate an excellent and stable neural adhesion and functional behavior comparable with what expected using a standard adhesion factor, such as polylysine or laminin, thus demonstrating that this procedure can be considered a good starting point to develop alternatives to the traditional chip coatings to provide selective and specific neuron-substrate adhesion.

Following the ontogeny of retinal waves: pan-retinal recordings of population dynamics in the neonatal mouse.

The immature retina generates spontaneous waves of spiking activity that sweep across the ganglion cell layer during a limited period of development before the onset of visual experience. The spatiotemporal patterns encoded in the waves are believed to be instructive for the wiring of functional connections throughout the visual system. However, the ontogeny of retinal waves is still poorly documented as a result of the relatively low resolution of conventional recording techniques. Here, we characterize the spatiotemporal features of mouse retinal waves from birth until eye opening in unprecedented detail using a large-scale, dense, 4096-channel multielectrode array that allowed us to record from the entire neonatal retina at near cellular resolution. We found that early cholinergic waves propagate with random trajectories over large areas with low ganglion cell recruitment. They become slower, smaller and denser when GABAA signalling matures, as occurs beyond postnatal day (P) 7. Glutamatergic influences dominate from P10, coinciding with profound changes in activity dynamics. At this time, waves cease to be random and begin to show repetitive trajectories confined to a few localized hotspots. These hotspots gradually tile the retina with time, and disappear after eye opening. Our observations demonstrate that retinal waves undergo major spatiotemporal changes during ontogeny. Our results support the hypotheses that cholinergic waves guide the refinement of retinal targets and that glutamatergic waves may also support the wiring of retinal receptive fields.

Ontogeny of the circadian system: a multiscale process throughout development.

The 24 h (circadian) timing system develops in mammals during the perinatal period. It carries out the essential task of anticipating daily recurring environmental changes to identify the best time of day for each molecular, cellular, and systemic process. Although significant knowledge has been acquired about the organization and function of the adult circadian system, relatively little is known about its ontogeny. During the perinatal period, the circadian system progressively gains functionality under the influence of the early environment. This review explores current evidence on the development of the circadian clock in mammals, highlighting the multilevel complexity of the process and the importance of gaining a better understanding of its underlying biology.

Advancing the interfacing performances of chronically implantable neural probes in the era of CMOS neuroelectronics.

Tissue penetrating microelectrode neural probes can record electrophysiological brain signals at resolutions down to single neurons, making them invaluable tools for neuroscience research and Brain-Computer-Interfaces (BCIs). The known gradual decrease of their electrical interfacing performances in chronic settings, however, remains a major challenge. A key factor leading to such decay is Foreign Body Reaction (FBR), which is the cascade of biological responses that occurs in the brain in the presence of a tissue damaging artificial device. Interestingly, the recent adoption of Complementary Metal Oxide Semiconductor (CMOS) technology to realize implantable neural probes capable of monitoring hundreds to thousands of neurons simultaneously, may open new opportunities to face the FBR challenge. Indeed, this shift from passive Micro Electro-Mechanical Systems (MEMS) to active CMOS neural probe technologies creates important, yet unexplored, opportunities to tune probe features such as the mechanical properties of the probe, its layout, size, and surface physicochemical properties, to minimize tissue damage and consequently FBR. Here, we will first review relevant literature on FBR to provide a better understanding of the processes and sources underlying this tissue response. Methods to assess FBR will be described, including conventional approaches based on the imaging of biomarkers, and more recent transcriptomics technologies. Then, we will consider emerging opportunities offered by the features of CMOS probes. Finally, we will describe a prototypical neural probe that may meet the needs for advancing clinical BCIs, and we propose axial insertion force as a potential metric to assess the influence of probe features on acute tissue damage and to control the implantation procedure to minimize iatrogenic injury and subsequent FBR.

Astrocytes actively support long-range molecular clock synchronization of segregated neuronal populations.

In mammals, the suprachiasmatic nucleus of the hypothalamus is the master circadian pacemaker that synchronizes the clocks in the central nervous system and periphery, thus orchestrating rhythms throughout the body. However, little is known about how so many cellular clocks within and across brain circuits can be effectively synchronized. In this work, we investigated the implication of two possible pathways: (i) astrocytes-mediated synchronization and (ii) neuronal paracrine factors-mediated synchronization. By taking advantage of a lab-on-a-chip microfluidic device developed in our laboratory, here we report that both pathways are involved. We found the paracrine factors-mediated synchronization of molecular clocks is diffusion-limited and, in our device, effective only in case of a short distance between neuronal populations. Interestingly, interconnecting astrocytes define an active signaling channel that can synchronize molecular clocks of neuronal populations also at longer distances. At mechanism level, we found that astrocytes-mediated synchronization involves both GABA and glutamate, while neuronal paracrine factors-mediated synchronization occurs through GABA signaling. These findings identify a previously unknown role of astrocytes as active cells that might distribute long-range signals to synchronize the brain clocks, thus further strengthening the importance of reciprocal interactions between glial and neuronal cells in the context of circadian circuitry.

Integrated Micro-Devices for a Lab-in-Organoid Technology Platform: Current Status and Future Perspectives.

Advancements in stem cell technology together with an improved understanding of in vitro organogenesis have enabled new routes that exploit cell-autonomous self-organization responses of adult stem cells (ASCs) and homogenous pluripotent stem cells (PSCs) to grow complex, three-dimensional (3D), mini-organ like structures on demand, the so-called organoids. Conventional optical and electrical neurophysiological techniques to acquire functional data from brain organoids, however, are not adequate for chronic recordings of neural activity from these model systems, and are not ideal approaches for throughput screenings applied to drug discovery. To overcome these issues, new emerging approaches aim at fusing sensing mechanisms and/or actuating artificial devices within organoids. Here we introduce and develop the concept of the Lab-in-Organoid (LIO) technology for in-tissue sensing and actuation within 3D cell aggregates. This challenging technology grounds on the self-aggregation of brain cells and on integrated bioelectronic micro-scale devices to provide an advanced tool for generating 3D biological brain models with in-tissue artificial functionalities adapted for routine, label-free functional measurements and for assay's development. We complete previously reported results on the implementation of the integrated self-standing wireless silicon micro-devices with experiments aiming at investigating the impact on neuronal spheroids of sinusoidal electro-magnetic fields as those required for wireless power and data transmission. Finally, we discuss the technology headway and future perspectives.

Glial Bmal1 role in mammalian retina daily changes.

Visual information processing in the retina requires the rhythmic expression of clock genes. The intrinsic retinal circadian clock is independent of the master clock located in the hypothalamic suprachiasmatic nucleus and emerges from retinal cells, including glia. Less clear is how glial oscillators influence the daily regulation of visual information processing in the mouse retina. Here, we demonstrate that the adult conditional deletion of the gene Bmal1 in GLAST-positive glial cells alters retinal physiology. Specifically, such deletion was sufficient to lower the amplitude of the electroretinogram b-wave recorded under light-adapted conditions. Furthermore, recordings from > 20,000 retinal ganglion cells (RGCs), the retina output, showed a non-uniform effect on RGCs activity in response to light across different cell types and over a 24-h period. Overall, our results suggest a new role of a glial circadian gene in adjusting mammalian retinal output throughout the night-day cycle.

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC-electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

Surface-Functionalized Self-Standing Microdevices Exhibit Predictive Localization and Seamless Integration in 3D Neural Spheroids.

Brain organoids is an exciting technology proposed to advance studies on human brain development, diseases, and possible therapies. Establishing and applying such models, however, is hindered by the lack of technologies to chronically monitor neural activity. A promising new approach comprising self-standing biosensing microdevices capable of achieving seamless tissue integration during cell aggregation and culture. To date, there is little information on how to control the aggregation of such bioartificial 3D neural assemblies. Here, the growth of hybrid neurospheroids obtained by the aggregation of silicon sham microchips (100 × 100 × 50 µm3 ) with primary cortical cells is investigated. Results obtained via protein-binding microchips with different molecules reveal that surface functionalization can tune the integration and final 3D location of self-standing microdevices into neurospheroids. Morphological and functional characterization suggests that the presence of an integrated microdevice does not alter spheroid growth, cellular composition, nor functional development. Ultimately, cells and microdevices constituting such hybrid neurospheroids can be disaggregated for further single-cell analysis, and quantifications confirm an unaltered ratio of neurons and glia. These results uncover the potential of surface-engineered self-standing microdevices to grow untethered 3D brain tissue models with inbuilt bioelectronic sensors at predefined sites.

Modeling a population of retinal ganglion cells with restricted Boltzmann machines.

The retina is a complex circuit of the central nervous system whose aim is to encode visual stimuli prior the higher order processing performed in the visual cortex. Due to the importance of its role, modeling the retina to advance in interpreting its spiking activity output is a well studied problem. In particular, it has been shown that latent variable models can be used to model the joint distribution of Retinal Ganglion Cells (RGCs). In this work, we validate the applicability of Restricted Boltzmann Machines to model the spiking activity responses of a large a population of RGCs recorded with high-resolution electrode arrays. In particular, we show that latent variables can encode modes in the RGC activity distribution that are closely related to the visual stimuli. In contrast to previous work, we further validate our findings by comparing results associated with recordings from retinas under normal and altered encoding conditions obtained by pharmacological manipulation. In these conditions, we observe that the model reflects well-known physiological behaviors of the retina. Finally, we show that we can also discover temporal patterns, associated with distinct dynamics of the stimuli.

Active pixel sensor array for high spatio-temporal resolution electrophysiological recordings from single cell to large scale neuronal networks.

This paper presents a chip-based electrophysiological platform enabling the study of micro- and macro-circuitry in in-vitro neuronal preparations. The approach is based on a 64x64 microelectrode array device providing extracellular electrophysiological activity recordings with high spatial (21 microm of electrode separation) and temporal resolution (from 0.13 ms for 4096 microelectrodes down to 8 micros for 64 microelectrodes). Applied to in-vitro neuronal preparations, we show how this approach enables neuronal signals to be acquired for investigating neuronal activity from single cells and microcircuits to large scale neuronal networks. The main elements of the platform are the metallic microelectrode array (MEA) implemented in Complementary Metal Oxide Semiconductor (CMOS) technology similar to a light imager, the in-pixel integrated low-noise amplifiers (11 microVrms) and the high-speed random addressing logic. The chip is combined with a real-time acquisition system providing the capability to record at 7.8 kHz/electrode the whole array and to process the acquired signals.

Astrocytes and Circadian Rhythms: An Emerging Astrocyte-Neuron Synergy in the Timekeeping System.

Animals have an internal timekeeping system to anticipate daily changes associated with the transition of day to night, which is deeply involved in the regulation and maintenance of behavioral and physiological processes. Prevailing knowledge associated the control of circadian clocks to a network of neurons in the central pacemaker, the suprachiasmatic nucleus (SCN), but astrocytes are rapidly emerging as key cellular contributors to the timekeeping system. However, how these glial cells impact the neuronal clock to modulate rhythmic neurobehavioral outputs just begin to be investigated. Astrocyte-neuron cocultures are an excellent exploratory method to further characterize the critical role of circadian communication between nerve cells, as well as to address the role of astrocytes as modulators and targets of neuronal rhythmic behaviors. Here, we describe a robust method to study astrocyte rhythmic interactions with neurons by coculturing them with primary neurons in physically separated layers. This simple coculture system provides hints on in vivo signaling processes. Moreover, it allows investigating cell-type specific effects separately as well as the identification of extracellular astrocytic or neuronal factors involved in rhythm generation in both cell types.

Investigating the Effects of Mechanical Stimulation on Retinal Ganglion Cell Spontaneous Spiking Activity.

Mechanical forces are increasingly recognized as major regulators of several physiological processes at both the molecular and cellular level; therefore, a deep understanding of the sensing of these forces and their conversion into electrical signals are essential for studying the mechanosensitive properties of soft biological tissues. To contribute to this field, we present a dual-purpose device able to mechanically stimulate retinal tissue and to record the spiking activity of retinal ganglion cells (RGCs). This new instrument relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with high-density multi-electrode array (HD-MEAs) to simultaneously record the spontaneous activity of the retina. In this paper, we introduce this instrument, describe its technical characteristics, and present a proof-of-concept experiment that shows how RGC spiking activity of explanted mice retinas respond to mechanical micro-stimulations of their photoreceptor layer. The data suggest that, under specific conditions of indentation, the retina perceive the mechanical stimulation as modulation of the visual input, besides the longer time-scale of activation, and the increase in spiking activity is not only localized under the indentation probe, but it propagates across the retinal tissue.

Active High-Density Electrode Arrays: Technology and Applications in Neuronal Cell Cultures.

Active high-density electrode arrays realized with complementary metal-oxide-semiconductor (CMOS) technology provide electrophysiological recordings from several thousands of closely spaced microelectrodes. This has drastically advanced the spatiotemporal recording resolution of conventional multielectrode arrays (MEAs). Thus, today's electrophysiology in neuronal cultures can exploit label-free electrical readouts from a large number of single neurons within the same network. This provides advanced capabilities to investigate the properties of self-assembling neuronal networks, to advance studies on neurotoxicity and neurodevelopmental alterations associated with human brain diseases, and to develop cell culture models for testing drug- or cell-based strategies for therapies.Here, after introducing the reader to this neurotechnology, we summarize the results of different recent studies demonstrating the potential of active high-density electrode arrays for experimental applications. We also discuss ongoing and possible future research directions that might allow for moving these platforms forward for screening applications.

SiNAPS: An implantable active pixel sensor CMOS-probe for simultaneous large-scale neural recordings.

Large-scale neural recordings with high spatial and temporal accuracy are instrumental to understand how the brain works. To this end, it is of key importance to develop probes that can be conveniently scaled up to a high number of recording channels. Despite recent achievements in complementary metal-oxide semiconductor (CMOS) multi-electrode arrays probes, in current circuit architectures an increase in the number of simultaneously recording channels would significantly increase the total chip area. A promising approach for overcoming this scaling issue consists in the use of the modular Active Pixel Sensor (APS) concept, in which a small front-end circuit is located beneath each electrode. However, this approach imposes challenging constraints on the area of the in-pixel circuit, power consumption and noise. Here, we present an APS CMOS-probe technology for Simultaneous Neural recording that successfully addresses all these issues for whole-array read-outs at 25 kHz/channel from up to 1024 electrode-pixels. To assess the circuit performances, we realized in a 0.18 µm CMOS technology an implantable single-shaft probe with a regular array of 512 electrode-pixels with a pitch of 28 µm. Extensive bench tests showed an in-pixel gain of 45.4 ± 0.4 dB (low pass, F-3 dB = 4 kHz), an input referred noise of 7.5 ± 0.67 µVRMS (300 Hz to 7.5 kHz) and a power consumption <6 µW/pixel. In vivo acute recordings demonstrate that our SiNAPS CMOS-probe can sample full-band bioelectrical signals from each electrode, with the ability to resolve and discriminate activity from several packed neurons both at the spatial and temporal scale. These results pave the way to new generations of compact and scalable active single/multi-shaft brain recording systems.

Biofunctionalized 3D Nanopillar Arrays Fostering Cell Guidance and Promoting Synapse Stability and Neuronal Activity in Networks.

A controlled geometry of in vitro neuronal networks allows investigation of the cellular mechanisms that underlie neuron-to-neuron and neuron-extracellular matrix interactions, which are essential to biomedical research. Herein, we report a selective guidance of primary hippocampal neurons by using arrays of three-dimensional vertical nanopillars (NPs) functionalized with a specific adhesion-promoting molecule-poly-dl-ornithine (PDLO). We show that 90% of neuronal cells are guided exclusively on the combinatorial PDLO/NP substrate. Moreover, we demonstrate the influence of the interplay between nanostructures and neurons on synapse formation and maturation, resulting in increased expression of postsynaptic density-95 protein and enhanced network cellular activity conferred by the endogenous c-fos expression. Successful guidance to foster synapse stability and cellular activity on multilevel cues of surface topography and chemical functionalization suggests the potential to devise technologies to control neuronal growth on nanostructures for tissue engineering, neuroprostheses, and drug development.

Fabrication of Multielectrode Arrays for Neurobiology Applications.

Substrate-integrated multielectrode arrays (MEAs) enable multisite, long-term, and label-free sensing and actuation of neuronal electrical signals in reduced cell culture models for network electrophysiology. Conventional, thin-film fabricated passive MEAs typically provide a few tens of electrode sites. New generations of active CMOS-based high-resolution arrays provide the capabilities of simultaneous recordings from thousands of neurons over fields of view of several square millimeters, yet allowing extracellular electrical imaging to be achieved down to the subcellular scale. In turn, such advancement in chip-based electrical readouts can significantly complement recently developed biotechnological and bimolecular techniques for neurobiology applications. Here, we describe (1) a simple method to fabricate passive MEAs and (2) protocols for preparing and growing primary rat hippocampal neuronal cultures and human iPS-derived neurons on MEAs. The aim is to provide reliable protocols for initiating the reader to this technology and for stimulating their further development and experimental use in neurobiology.