A live mammalian cells electroporation array for on-chip immunofluorescence.

The detection of intracellular proteins in vitro is commonly realized with immunofluorescence techniques, through which antibodies or markers are delivered into fixed cells and recognize specific proteins. Many innovative techniques, however, avoid cells fixation by chemical compounds and, among the others, electroporation is widely used. Here we demonstrate that in situ electroporation on thin film SiO2 capacitive microelectrodes can be realized with high efficiency to deliver fluorescent markers and antibodies into mammalian cell lines and primary neuronal cells to detect intracellular proteins, like actin. The results presented in this work open the way to the use of this technique for the detection of potentially any target protein, even through subsequent electroporations.

Early Detection of Alzheimer's Disease From Cortical and Hippocampal Local Field Potentials Using an Ensembled Machine Learning Model.

Early diagnosis of Alzheimer's disease (AD) is a very challenging problem and has been attempted through data-driven methods in recent years. However, considering the inherent complexity in decoding higher cognitive functions from spontaneous neuronal signals, these data-driven methods benefit from the incorporation of multimodal data. This work proposes an ensembled machine learning model with explainability (EXML) to detect subtle patterns in cortical and hippocampal local field potential signals (LFPs) that can be considered as a potential marker for AD in the early stage of the disease. The LFPs acquired from healthy and two types of AD animal models (n = 10 each) using linear multielectrode probes were endorsed by electrocardiogram and respiration signals for their veracity. Feature sets were generated from LFPs in temporal, spatial and spectral domains and were fed into selected machine-learning models for each domain. Using late fusion, the EXML model achieved an overall accuracy of 99.4%. This provided insights into the amyloid plaque deposition process as early as 3 months of the disease onset by identifying the subtle patterns in the network activities. Lastly, the individual and ensemble models were found to be robust when evaluated by randomly masking channels to mimic the presence of artefacts.

Interfacing Biology and Electronics with Memristive Materials.

Memristive technologies promise to have a large impact on modern electronics, particularly in the areas of reconfigurable computing and artificial intelligence (AI) hardware. Meanwhile, the evolution of memristive materials alongside the technological progress is opening application perspectives also in the biomedical field, particularly for implantable and lab-on-a-chip devices where advanced sensing technologies generate a large amount of data. Memristive devices are emerging as bioelectronic links merging biosensing with computation, acting as physical processors of analog signals or in the framework of advanced digital computing architectures. Recent developments in the processing of electrical neural signals, as well as on transduction and processing of chemical biomarkers of neural and endocrine functions, are reviewed. It is concluded with a critical perspective on the future applicability of memristive devices as pivotal building blocks in bio-AI fusion concepts and bionic schemes.

Disentangling the critical signatures of neural activity.

The critical brain hypothesis has emerged as an attractive framework to understand neuronal activity, but it is still widely debated. In this work, we analyze data from a multi-electrodes array in the rat's cortex and we find that power-law neuronal avalanches satisfying the crackling-noise relation coexist with spatial correlations that display typical features of critical systems. In order to shed a light on the underlying mechanisms at the origin of these signatures of criticality, we introduce a paradigmatic framework with a common stochastic modulation and pairwise linear interactions inferred from our data. We show that in such models power-law avalanches that satisfy the crackling-noise relation emerge as a consequence of the extrinsic modulation, whereas scale-free correlations are solely determined by internal interactions. Moreover, this disentangling is fully captured by the mutual information in the system. Finally, we show that analogous power-law avalanches are found in more realistic models of neural activity as well, suggesting that extrinsic modulation might be a broad mechanism for their generation.

Classification of Whisker Deflections From Evoked Responses in the Somatosensory Barrel Cortex With Spiking Neural Networks.

Spike-based neuromorphic hardware has great potential for low-energy brain-machine interfaces, leading to a novel paradigm for neuroprosthetics where spiking neurons in silicon read out and control activity of brain circuits. Neuromorphic processors can receive rich information about brain activity from both spikes and local field potentials (LFPs) recorded by implanted neural probes. However, it was unclear whether spiking neural networks (SNNs) implemented on such devices can effectively process that information. Here, we demonstrate that SNNs can be trained to classify whisker deflections of different amplitudes from evoked responses in a single barrel of the rat somatosensory cortex. We show that the classification performance is comparable or even superior to state-of-the-art machine learning approaches. We find that SNNs are rather insensitive to recorded signal type: both multi-unit spiking activity and LFPs yield similar results, where LFPs from cortical layers III and IV seem better suited than those of deep layers. In addition, no hand-crafted features need to be extracted from the data-multi-unit activity can directly be fed into these networks and a simple event-encoding of LFPs is sufficient for good performance. Furthermore, we find that the performance of SNNs is insensitive to the network state-their performance is similar during UP and DOWN states.

Increased fMRI connectivity upon chemogenetic inhibition of the mouse prefrontal cortex.

While shaped and constrained by axonal connections, fMRI-based functional connectivity reorganizes in response to varying interareal input or pathological perturbations. However, the causal contribution of regional brain activity to whole-brain fMRI network organization remains unclear. Here we combine neural manipulations, resting-state fMRI and in vivo electrophysiology to probe how inactivation of a cortical node causally affects brain-wide fMRI coupling in the mouse. We find that chronic inhibition of the medial prefrontal cortex (PFC) via overexpression of a potassium channel increases fMRI connectivity between the inhibited area and its direct thalamo-cortical targets. Acute chemogenetic inhibition of the PFC produces analogous patterns of fMRI overconnectivity. Using in vivo electrophysiology, we find that chemogenetic inhibition of the PFC enhances low frequency (0.1-4 Hz) oscillatory power via suppression of neural firing not phase-locked to slow rhythms, resulting in increased slow and δ band coherence between areas that exhibit fMRI overconnectivity. These results provide causal evidence that cortical inactivation can counterintuitively increase fMRI connectivity via enhanced, less-localized slow oscillatory processes.

Accelerated Aging Characterizes the Early Stage of Alzheimer's Disease.

For Alzheimer's disease (AD), aging is the main risk factor, but whether cognitive impairments due to aging resemble early AD deficits is not yet defined. When working with mouse models of AD, the situation is just as complicated, because only a few studies track the progression of the disease at different ages, and most ignore how the aging process affects control mice. In this work, we addressed this problem by comparing the aging process of PS2APP (AD) and wild-type (WT) mice at the level of spontaneous brain electrical activity under anesthesia. Using local field potential recordings, obtained with a linear probe that traverses the posterior parietal cortex and the entire hippocampus, we analyzed how multiple electrical parameters are modified by aging in AD and WT mice. With this approach, we highlighted AD specific features that appear in young AD mice prior to plaque deposition or that are delayed at 12 and 16 months of age. Furthermore, we identified aging characteristics present in WT mice but also occurring prematurely in young AD mice. In short, we found that reduction in the relative power of slow oscillations (SO) and Low/High power imbalance are linked to an AD phenotype at its onset. The loss of SO connectivity and cortico-hippocampal coupling between SO and higher frequencies as well as the increase in UP-state and burst durations are found in young AD and old WT mice. We show evidence that the aging process is accelerated by the mutant PS2 itself and discuss such changes in relation to amyloidosis and gliosis.

Simultaneous Two-Photon Voltage or Calcium Imaging and Multi-Channel Local Field Potential Recordings in Barrel Cortex of Awake and Anesthetized Mice.

Neuronal population activity, both spontaneous and sensory-evoked, generates propagating waves in cortex. However, high spatiotemporal-resolution mapping of these waves is difficult as calcium imaging, the work horse of current imaging, does not reveal subthreshold activity. Here, we present a platform combining voltage or calcium two-photon imaging with multi-channel local field potential (LFP) recordings in different layers of the barrel cortex from anesthetized and awake head-restrained mice. A chronic cranial window with access port allows injecting a viral vector expressing GCaMP6f or the voltage-sensitive dye (VSD) ANNINE-6plus, as well as entering the brain with a multi-channel neural probe. We present both average spontaneous activity and average evoked signals in response to multi-whisker air-puff stimulations. Time domain analysis shows the dependence of the evoked responses on the cortical layer and on the state of the animal, here separated into anesthetized, awake but resting, and running. The simultaneous data acquisition allows to compare the average membrane depolarization measured with ANNINE-6plus with the amplitude and shape of the LFP recordings. The calcium imaging data connects these data sets to the large existing database of this important second messenger. Interestingly, in the calcium imaging data, we found a few cells which showed a decrease in calcium concentration in response to vibrissa stimulation in awake mice. This system offers a multimodal technique to study the spatiotemporal dynamics of neuronal signals through a 3D architecture in vivo. It will provide novel insights on sensory coding, closing the gap between electrical and optical recordings.

Neuronal Avalanches Across the Rat Somatosensory Barrel Cortex and the Effect of Single Whisker Stimulation.

Since its first experimental signatures, the so called "critical brain hypothesis" has been extensively studied. Yet, its actual foundations remain elusive. According to a widely accepted teleological reasoning, the brain would be poised to a critical state to optimize the mapping of the noisy and ever changing real-world inputs, thus suggesting that primary sensory cortical areas should be critical. We investigated whether a single barrel column of the somatosensory cortex of the anesthetized rat displays a critical behavior. Neuronal avalanches were recorded across all cortical layers in terms of both multi-unit activities and population local field potentials, and their behavior during spontaneous activity compared to the one evoked by a controlled single whisker deflection. By applying a maximum likelihood statistical method based on timeseries undersampling to fit the avalanches distributions, we show that neuronal avalanches are power law distributed for both multi-unit activities and local field potentials during spontaneous activity, with exponents that are spread along a scaling line. Instead, after the tactile stimulus, activity switches to a transient across-layers synchronization mode that appears to dominate the cortical representation of the single sensory input.

Understanding the Effects of Anesthesia on Cortical Electrophysiological Recordings: A Scoping Review.

General anesthesia in animal experiments is an ethical must and is required for all the procedures that are likely to cause more than slight or momentary pain. As anesthetics are known to deeply affect experimental findings, including electrophysiological recordings of brain activity, understanding their mechanism of action is of paramount importance. It is widely recognized that the depth and type of anesthesia introduce significant bias in electrophysiological measurements by affecting the shape of both spontaneous and evoked signals, e.g., modifying their latency and relative amplitude. Therefore, for a given experimental protocol, it is relevant to identify the appropriate anesthetic, to minimize the impact on neuronal circuits and related signals under investigation. This review focuses on the effect of different anesthetics on cortical electrical recordings, examining their molecular mechanisms of action, their influence on neuronal microcircuits and, consequently, their impact on cortical measurements.

Plasticity and Adaptation in Neuromorphic Biohybrid Systems.

Neuromorphic systems take inspiration from the principles of biological information processing to form hardware platforms that enable the large-scale implementation of neural networks. The recent years have seen both advances in the theoretical aspects of spiking neural networks for their use in classification and control tasks and a progress in electrophysiological methods that is pushing the frontiers of intelligent neural interfacing and signal processing technologies. At the forefront of these new technologies, artificial and biological neural networks are tightly coupled, offering a novel "biohybrid" experimental framework for engineers and neurophysiologists. Indeed, biohybrid systems can constitute a new class of neuroprostheses opening important perspectives in the treatment of neurological disorders. Moreover, the use of biologically plausible learning rules allows forming an overall fault-tolerant system of co-developing subsystems. To identify opportunities and challenges in neuromorphic biohybrid systems, we discuss the field from the perspectives of neurobiology, computational neuroscience, and neuromorphic engineering.

Author Correction: Memristive synapses connect brain and silicon spiking neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Memristive synapses connect brain and silicon spiking neurons.

Brain function relies on circuits of spiking neurons with synapses playing the key role of merging transmission with memory storage and processing. Electronics has made important advances to emulate neurons and synapses and brain-computer interfacing concepts that interlink brain and brain-inspired devices are beginning to materialise. We report on memristive links between brain and silicon spiking neurons that emulate transmission and plasticity properties of real synapses. A memristor paired with a metal-thin film titanium oxide microelectrode connects a silicon neuron to a neuron of the rat hippocampus. Memristive plasticity accounts for modulation of connection strength, while transmission is mediated by weighted stimuli through the thin film oxide leading to responses that resemble excitatory postsynaptic potentials. The reverse brain-to-silicon link is established through a microelectrode-memristor pair. On these bases, we demonstrate a three-neuron brain-silicon network where memristive synapses undergo long-term potentiation or depression driven by neuronal firing rates.

Dampened Slow Oscillation Connectivity Anticipates Amyloid Deposition in the PS2APP Mouse Model of Alzheimer's Disease.

To fight Alzheimer's disease (AD), we should know when, where, and how brain network dysfunctions initiate. In AD mouse models, relevant information can be derived from brain electrical activity. With a multi-site linear probe, we recorded local field potentials simultaneously at the posterior-parietal cortex and hippocampus of wild-type and double transgenic AD mice, under anesthesia. We focused on PS2APP (B6.152H) mice carrying both presenilin-2 (PS2) and amyloid precursor protein (APP) mutations, at three and six months of age, before and after plaque deposition respectively. To highlight defects linked to either the PS2 or APP mutation, we included in the analysis age-matched PS2.30H and APP-Swedish mice, carrying each of the mutations individually. Our study also included PSEN2-/- mice. At three months, only predeposition B6.152H mice show a reduction in the functional connectivity of slow oscillations (SO) and in the power ratio between SO and delta waves. At six months, plaque-seeding B6.152H mice undergo a worsening of the low/high frequency power imbalance and show a massive loss of cortico-hippocampal phase-amplitude coupling (PAC) between SO and higher frequencies, a feature shared with amyloid-free PS2.30H mice. We conclude that the PS2 mutation is sufficient to impair SO PAC and accelerate network dysfunctions in amyloid-accumulating mice.

Open-Source Tools for Processing and Analysis of In Vitro Extracellular Neuronal Signals.

The recent years have seen unprecedented growth in the manufacturing of neurotechnological tools. The latest technological advancements presented the neuroscientific community with neuronal probes containing thousands of recording sites. These next-generation probes are capable of simultaneously recording neuronal signals from a large number of channels. Numerically, a simple 128-channel neuronal data acquisition system equipped with a 16 bits A/D converter digitizing the acquired analog waveforms at a sampling frequency of 20 kHz will generate approximately 17 GB uncompressed data per hour. Today's biggest challenge is to mine this staggering amount of data and find useful information which can later be used in decoding brain functions, diagnosing diseases, and devising treatments. To this goal, many automated processing and analysis tools have been developed and reported in the literature. A good amount of them are also available as open source for others to adapt them to individual needs. Focusing on extracellularly recorded neuronal signals in vitro, this chapter provides an overview of the popular open-source tools applicable on these signals for spike trains and local field potentials analysis, and spike sorting. Towards the end, several future research directions have also been outlined.

Applications of Deep Learning and Reinforcement Learning to Biological Data.

Rapid advances in hardware-based technologies during the past decades have opened up new possibilities for life scientists to gather multimodal data in various application domains, such as omics, bioimaging, medical imaging, and (brain/body)-machine interfaces. These have generated novel opportunities for development of dedicated data-intensive machine learning techniques. In particular, recent research in deep learning (DL), reinforcement learning (RL), and their combination (deep RL) promise to revolutionize the future of artificial intelligence. The growth in computational power accompanied by faster and increased data storage, and declining computing costs have already allowed scientists in various fields to apply these techniques on data sets that were previously intractable owing to their size and complexity. This paper provides a comprehensive survey on the application of DL, RL, and deep RL techniques in mining biological data. In addition, we compare the performances of DL techniques when applied to different data sets across various application domains. Finally, we outline open issues in this challenging research area and discuss future development perspectives.

Sub 100 nW Volatile Nano-Metal-Oxide Memristor as Synaptic-Like Encoder of Neuronal Spikes.

Advanced neural interfaces mediate a bioelectronic link between the nervous system and microelectronic devices, bearing great potential as innovative therapy for various diseases. Spikes from a large number of neurons are recorded leading to creation of big data that require online processing under most stringent conditions, such as minimal power dissipation and on-chip space occupancy. Here, we present a new concept where the inherent volatile properties of a nano-scale memristive device are used to detect and compress information on neural spikes as recorded by a multielectrode array. Simultaneously, and similarly to a biological synapse, information on spike amplitude and frequency is transduced in metastable resistive state transitions of the device, which is inherently capable of self-resetting and of continuous encoding of spiking activity. Furthermore, operating the memristor in a very high resistive state range reduces its average in-operando power dissipation to less than 100 nW, demonstrating the potential to build highly scalable, yet energy-efficient on-node processors for advanced neural interfaces.

Algorithm and software to automatically identify latency and amplitude features of local field potentials recorded in electrophysiological investigation.

BACKGROUND: Local field potentials (LFPs) evoked by sensory stimulation are particularly useful in electrophysiological research. For instance, spike timing and current transmembrane current flow estimated from LFPs recorded in the barrel cortex in rats and mice are exploited to investigate how the brain represents sensory stimuli. Recent improvements in microelectrodes technology enable neuroscientists to acquire a great amount of LFPs during the same experimental session, calling for algorithms for their quantitative automatic analysis. Several computer tools were proposed for LFP analysis, but many of them incorporate algorithms that are not open to inspection or modification/personalization. We present a MATLAB software to automatically detect some important LFP features (latency, amplitude, time-derivative value in the inflection-point) for a quantitative analysis. The software features can be customized by the user according to his/her personal research needs. The incorporated algorithm is based on Phillips-Tikhonov regularization to deal with noise amplification due to ill-conditioning. In particular, its accuracy in the estimation of the features of interest is assessed in a Monte Carlo simulation mimicking the acquisition of LFPs in different SNR (signal-to-noise-ratio) conditions. Then, the algorithm is tested by analyzing a real set of 2500 LFPs recorded in rat after whisker stimulation at different depths in the primary somatosensory (S1) cortex, i.e., the region involved in the cortical representation of touch in mammals. RESULTS: Automatic identification of LFP features by the presented software is easy and fast. As far as accuracy is concerned, error indices from simulated data suggest that the algorithm provides reliable estimates . Indeed, results obtained from LFPs recorded in rat after whisker stimulation are in line with the known sequential activation of the microcircuits of the S1 cortex. CONCLUSION: A MATLAB software implementing an algorithm to automatically detect the main LFPs features was presented. Simulated and real case studies showed that the employed algorithm is accurate and robust against measurement noise. The available code can be used as it is, but the reported description of the algorithms allows users to easily modify the code to cope with specific requirements.

Intranasal Oxytocin and Vasopressin Modulate Divergent Brainwide Functional Substrates.

The neuropeptides oxytocin (OXT) and vasopressin (AVP) have been identified as modulators of emotional social behaviors and associated with neuropsychiatric disorders characterized by social dysfunction. Experimental and therapeutic use of OXT and AVP via the intranasal route is the subject of extensive clinical research. However, the large-scale functional substrates directly engaged by these peptides and their functional dynamics remain elusive. By using cerebral blood volume (CBV) weighted fMRI in the mouse, we show that intranasal administration of OXT rapidly elicits the transient activation of cortical regions and a sustained activation of hippocampal and forebrain areas characterized by high oxytocin receptor density. By contrast, intranasal administration of AVP produced a robust and sustained deactivation in cortico-parietal, thalamic and mesolimbic regions. Importantly, intravenous administration of OXT and AVP did not recapitulate the patterns of modulation produced by intranasal dosing, supporting a central origin of the observed functional changes. In keeping with this notion, hippocampal local field potential recordings revealed multi-band power increases upon intranasal OXT administration. We also show that the selective OXT-derivative TGOT reproduced the pattern of activation elicited by OXT and that the deletion of OXT receptors does not affect AVP-mediated deactivation. Collectively, our data document divergent modulation of brainwide neural systems by intranasal administration of OXT and AVP, an effect that involves key substrates of social and emotional behavior. The observed divergence calls for a deeper investigation of the systems-level mechanisms by which exogenous OXT and AVP modulate brain function and exert their putative therapeutic effects.

Early hippocampal hyperexcitability in PS2APP mice: role of mutant PS2 and APP.

Alterations of brain network activity are observable in Alzheimer's disease (AD) together with the occurrence of mild cognitive impairment, before overt pathology. However, in humans as well in AD mouse models, identification of early biomarkers of network dysfunction is still at its beginning. We performed in vivo recordings of local field potential activity in the dentate gyrus of PS2APP mice expressing the human amyloid precursor protein (APP) Swedish mutation and the presenilin-2 (PS2) N141I. From a frequency-domain analysis, we uncovered network hyper-synchronicity as early as 3 months, when intracellular accumulation of amyloid beta was also observable. In addition, at 6 months of age, we identified network hyperactivity in the beta/gamma frequency bands, along with increased theta-beta and theta-gamma phase-amplitude cross-frequency coupling, in coincidence with the histopathological traits of the disease. Although hyperactivity and hypersynchronicity were respectively detected in mice expressing the PS2-N141I or the APP Swedish mutant alone, the increase in cross-frequency coupling specifically characterized the 6-month-old PS2APP mice, just before the surge of the cognitive decline.