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.

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.

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.

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.

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.

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.

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.

Exploiting All Programmable SoCs in Neural Signal Analysis: A Closed-Loop Control for Large-Scale CMOS Multielectrode Arrays.

Microelectrode array (MEA) systems with up to several thousands of recording electrodes and electrical or optical stimulation capabilities are commercially available or described in the literature. By exploiting their submillisecond and micrometric temporal and spatial resolutions to record bioelectrical signals, such emerging MEA systems are increasingly used in neuroscience to study the complex dynamics of neuronal networks and brain circuits. However, they typically lack the capability of implementing real-time feedback between the detection of neuronal spiking events and stimulation, thus restricting large-scale neural interfacing to open-loop conditions. In order to exploit the potential of such large-scale recording systems and stimulation, we designed and validated a fully reconfigurable FPGA-based processing system for closed-loop multichannel control. By adopting a Xilinx Zynq-all-programmable system on chip that integrates reconfigurable logic and a dual-core ARM-based processor on the same device, the proposed platform permits low-latency preprocessing (filtering and detection) of spikes acquired simultaneously from several thousands of electrode sites. To demonstrate the proposed platform, we tested its performances through ex vivo experiments on the mice retina using a state-of-the-art planar high-density MEA that samples 4096 electrodes at 18 kHz and record light-evoked spikes from several thousands of retinal ganglion cells simultaneously. Results demonstrate that the platform is able to provide a total latency from whole-array data acquisition to stimulus generation below 2 ms. This opens the opportunity to design closed-loop experiments on neural systems and biomedical applications using emerging generations of planar or implantable large-scale MEA systems.

A Synchronous Neural Recording Platform for Multiple High-Resolution CMOS Probes and Passive Electrode Arrays.

Electrophysiological signals in the brain are distributed over broad spatial and temporal scales. Monitoring these signals at multiple scales is fundamental in order to decipher how brain circuits operate and might dysfunction in disease. A possible strategy to enlarge the experimentally accessible spatial and temporal scales consists in combining the use of multiple probes with different resolutions and sensing areas. Here, we propose a neural recording system capable of simultaneous and synchronous acquisitions from a new generation of high-resolution CMOS probes (512 microelectrodes, 25 kHz/electrode whole-array sampling frequency) as well as from a custom-designed CMOS-based headstage. While CMOS probes can provide recordings from a large number of closely spaced electrodes on single-shaft devices, the CMOS-based headstage can be used to interface the wide range of available intra- or epi-cortical passive electrode array devices. The current platform was designed to simultaneously manage high-resolution recordings from up to four differently located CMOS probes and from a single 36-channels low-resolution passive electrode array device. The design, implementation, and performances for both ICs and for the FPGA-based interface are presented. Experiments on retina and neuronal culture preparations demonstrate the recording of neural spiking activity for both CMOS devices and the functionality of the system.

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.

State-dependent representation of stimulus-evoked activity in high-density recordings of neural cultures.

Neuronal responses to external stimuli vary from trial to trial partly because they depend on continuous spontaneous variations of the state of neural circuits, reflected in variations of ongoing activity prior to stimulus presentation. Understanding how post-stimulus responses relate to the pre-stimulus spontaneous activity is thus important to understand how state dependence affects information processing and neural coding, and how state variations can be discounted to better decode single-trial neural responses. Here we exploited high-resolution CMOS electrode arrays to record simultaneously from thousands of electrodes in in-vitro cultures stimulated at specific sites. We used information-theoretic analyses to study how ongoing activity affects the information that neuronal responses carry about the location of the stimuli. We found that responses exhibited state dependence on the time between the last spontaneous burst and the stimulus presentation and that the dependence could be described with a linear model. Importantly, we found that a small number of selected neurons carry most of the stimulus information and contribute to the state-dependent information gain. This suggests that a major value of large-scale recording is that it individuates the small subset of neurons that carry most information and that benefit the most from knowledge of its state dependence.

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.

Developmental excitatory-to-inhibitory GABA-polarity switch is disrupted in 22q11.2 deletion syndrome: a potential target for clinical therapeutics.

Individuals with 22q11.2 microdeletion syndrome (22q11.2 DS) show cognitive and behavioral dysfunctions, developmental delays in childhood and risk of developing schizophrenia and autism. Despite extensive previous studies in adult animal models, a possible embryonic root of this syndrome has not been determined. Here, in neurons from a 22q11.2 DS mouse model (Lgdel +/-), we found embryonic-premature alterations in the neuronal chloride cotransporters indicated by dysregulated NKCC1 and KCC2 protein expression levels. We demonstrate with large-scale spiking activity recordings a concurrent deregulation of the spontaneous network activity and homeostatic network plasticity. Additionally, Lgdel +/- networks at early development show abnormal neuritogenesis and void of synchronized spontaneous activity. Furthermore, parallel experiments on Dgcr8 +/- mouse cultures reveal a significant, yet not exclusive contribution of the dgcr8 gene to our phenotypes of Lgdel +/- networks. Finally, we show that application of bumetanide, an inhibitor of NKCC1, significantly decreases the hyper-excitable action of GABAA receptor signaling and restores network homeostatic plasticity in Lgdel +/- networks. Overall, by exploiting an on-a-chip 22q11.2 DS model, our results suggest a delayed GABA-switch in Lgdel +/- neurons, which may contribute to a delayed embryonic development. Prospectively, acting on the GABA-polarity switch offers a potential target for 22q11.2 DS therapeutic intervention.

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.

Selective Targeting of Neurons with Inorganic Nanoparticles: Revealing the Crucial Role of Nanoparticle Surface Charge.

Nanoparticles (NPs) are increasingly used in biomedical applications, but the factors that influence their interactions with living cells need to be elucidated. Here, we reveal the role of NP surface charge in determining their neuronal interactions and electrical responses. We discovered that negatively charged NPs administered at low concentration (10 nM) interact with the neuronal membrane and at the synaptic cleft, whereas positively and neutrally charged NPs never localize on neurons. This effect is shape and material independent. The presence of negatively charged NPs on neuronal cell membranes influences the excitability of neurons by causing an increase in the amplitude and frequency of spontaneous postsynaptic currents at the single cell level and an increase of both the spiking activity and synchronous firing at neural network level. The negatively charged NPs exclusively bind to excitable neuronal cells, and never to nonexcitable glial cells. This specific interaction was also confirmed by manipulating the electrophysiological activity of neuronal cells. Indeed, the interaction of negatively charged NPs with neurons is either promoted or hindered by pharmacological suppression or enhancement of the neuronal activity with tetrodotoxin or bicuculline, respectively. We further support our main experimental conclusions by using numerical simulations. This study demonstrates that negatively charged NPs modulate the excitability of neurons, revealing the potential use of NPs for controlling neuron activity.