design-surface-treatment-cellular-plating-culturing-modular-neuronal

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Tel-Aviv University

Information processing in the nervous system is based on parallel computation, adaptation and learning. These features cannot be easily implemented on conventional silicon devices. In order to obtain a better insight of how neurons process information, we have explored the possibility of using biological neurons as parallel and adaptable computing elements for image processing and pattern recognition. Commercially available multielectrode arrays (MEAs) were used to record and stimulate the electrical activity from neuronal cultures. By mapping digital images, i.e., arrays of pixels, into the stimulation of neuronal cultures, a low and bandpass filtering of images could be quickly and easily obtained. Responses to specific spatial patterns of stimulation were potentiated by an appropriate training (tetanization). Learning allowed pattern recognition and extraction of spatial features in processed images. Therefore, neurocomputers, (i.e., hybrid devices containing man-made elements and natural neurons) seem feasible and may become a new generation of computing devices, to be developed by a synergy of Neuroscience and Material Science.

Toward the neurocomputer: image processing and pattern recognition with neuronal cultures.

Information processing and coding were analysed in dissociated hippocampal cultures, grown on multielectrode arrays. Multisite stimulation was used to activate different neurons and pathways of the network. The neural activity was binned into firing rates and the variability of the firing of individual neurons and of the whole population was analysed. In individual neurons, the timing of the first action potential (AP) was rather precise from trial to trial, whereas the timing of later APs was much more variable. Pooling APs in an ensemble of neurons reduced the variability of the response and allowed stimuli varying in intensity to be distinguished reliably in a single trial. A similar decrease of variability was observed pooling the first evoked APs in an ensemble of neurons. The size of the neuronal pool (approximately 50-100 neurons) and the time bin (approximately 20 ms) necessary to provide reproducible responses are remarkably similar to those obtained in in vivo preparations and in small nervous systems. Blockage of excitatory synaptic pathways mediated by NMDA receptors improved the mutual information between the evoked response and stimulus properties. When inhibitory GABAergic pathways were blocked by bicuculline the opposite effect was obtained. These results show how ensemble averages and an appropriate balance between inhibition and excitation allow neuronal networks to process information in a fast and reliable way.

Statistical properties of information processing in neuronal networks.

Embryonic stem (ES) cells provide a flexible and unlimited source for a variety of neuronal types. Because mature neurons establish neuronal networks very easily, we tested whether ES-derived neurons are capable of generating functional networks and whether these networks, generated in vitro, are capable of processing information. Single-cell electrophysiology with pharmacological antagonists demonstrated the presence of both excitatory and inhibitory synaptic connections. Extracellular recording with planar multielectrode arrays showed that spontaneous bursts of electrical activity are present in ES-derived networks with properties remarkably similar to those of hippocampal neurons. When stimulated with extracellular electrodes, ES-derived neurons fired action potentials, and the evoked electrical activity spread throughout the culture. A statistical analysis indicated that ES-derived networks discriminated between stimuli of different intensity at a single trial level, a key feature for an efficient information processing. Thus, ES-derived neurons provide a novel in vitro strategy to create functional networks with defined computational properties.

Embryonic stem cell-derived neurons form functional networks in vitro.

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple--precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L(-1), pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.

Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence.

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics.

On the dynamics of the spontaneous activity in neuronal networks.

Developing cortical networks generate a variety of coherent activity patterns that participate in circuit refinement. Early network oscillations (ENOs) are the dominant network pattern in the rodent neocortex for a short period after birth. These large-scale calcium waves were shown to be largely driven by glutamatergic synapses albeit GABA is a major excitatory neurotransmitter in the cortex at such early stages, mediating synapse-driven giant depolarizing potentials (GDPs) in the hippocampus. Using functional multineuron calcium imaging together with single-cell and field potential recordings to clarify distinct network dynamics in rat cortical slices, we now report that the developing somatosensory cortex generates first ENOs then GDPs, both patterns coexisting for a restricted time period. These patterns markedly differ by their developmental profile, dynamics, and mechanisms: ENOs are generated before cortical GDPs (cGDPs) by the activation of glutamatergic synapses mostly through NMDARs; cENOs are low-frequency oscillations (approximately 0.01 Hz) displaying slow kinetics and gradually involving the entire network. At the end of the first postnatal week, GABA-driven cortical GDPs can be reliably monitored; cGDPs are recurrent oscillations (approximately 0.1 Hz) that repetitively synchronize localized neuronal assemblies. Contrary to cGDPs, cENOs were unexpectedly facilitated by short anoxic conditions suggesting a contribution of glutamate accumulation to their generation. In keeping with this, alterations of extracellular glutamate levels significantly affected cENOs, which are blocked by an enzymatic glutamate scavenger. Moreover, we show that a tonic glutamate current contributes to the neuronal membrane excitability when cENOs dominate network patterns. Therefore, cENOs and cGDPs are two separate aspects of neocortical network maturation that may be differentially engaged in physiological and pathological processes.

Sequential generation of two distinct synapse-driven network patterns in developing neocortex.

Structure-function studies of neuronal networks have recently benefited from considerable progress in different areas of investigation. Advances in molecular genetics and imaging have allowed for the dissection of neuronal connectivity with unprecedented detail whereas in vivo recordings are providing much needed clues as to how sensory, motor and cognitive function is encoded in neuronal firing. However, bridging the gap between the cellular and behavioral levels will ultimately require an understanding of the functional organization of the underlying neuronal circuits. One way to unravel the complexity of neuronal networks is to understand how their connectivity emerges during brain maturation. In this review, we will describe how graph theory provides experimentalists with novel concepts that can be used to describe and interpret these developing connectivity schemes.

Dissecting functional connectivity of neuronal microcircuits: experimental and theoretical insights.

Connectivity in the developing hippocampus displays a functional organization particularly effective in supporting network synchronization, as it includes superconnected hub neurons. We have previously shown that hub network function is supported by a subpopulation of GABA neurons. However, it is unclear whether hub cells are only transiently present or later develop into distinctive subclasses of interneurons. These questions are difficult to assess given the heterogeneity of the GABA neurons and the poor early expression of markers. To circumvent this conundrum, we used "genetic fate mapping" that allows for the selective labeling of GABA neurons based on their place and time of origin. We show that early-generated GABA cells form a subpopulation of hub neurons, characterized by an exceptionally widespread axonal arborization and the ability to single-handedly impact network dynamics when stimulated. Pioneer hub neurons remain into adulthood, when they acquire the classical markers of long-range projecting GABA neurons.

Pioneer GABA cells comprise a subpopulation of hub neurons in the developing hippocampus.

The growing recognition that brain pathologies do not affect neurons only but rather are, to a large extent, pathologies of glial cells as well as of the vasculature opens to new perspectives in our understanding of genetic disorders of the CNS. To validate the role of the neuron-glial-vascular unit in the etiology of genome instability disorders, we report about cell death and morphological aspects of neuroglia networks and the associated vasculature in a mouse model of Ataxia Telangiectasia (A-T), a human genetic disorder that induces severe motor impairment. We found that A-T-mutated protein deficiency was consistent with aberrant astrocytic morphology and alterations of the vasculature, often accompanied by reactive gliosis. Interestingly similar findings could also be reported in the case of other genetic disorders. These observations bolster the notion that astrocyte-specific pathologies, hampered vascularization and astrocyte-endothelium interactions in the CNS could play a crucial role in the etiology of genome instability brain disorders and could underlie neurodegeneration.

The role of the neuro-astro-vascular unit in the etiology of ataxia telangiectasia.

The developing CA3 hippocampus is comprised by highly connected hub neurons that are particularly effective in achieving network synchronization. Functional hub neurons were shown to be exclusively GABAergic, suggesting that the contribution of glutamatergic neurons to physiological synchronization processes at early postnatal stages is minimal. However, without fast GABAergic transmission, a different situation may prevail. In the adult CA3, blocking fast GABAergic transmission induces the generation of network bursts that can be triggered by the stimulation of single pyramidal neurons. Here we revisit the network function of CA3 glutamatergic neurons from a developmental viewpoint, without fast GABAergic transmission. We uncover a sub-population of early-generated glutamatergic neurons that impacts network dynamics when stimulated in the juvenile hippocampus. Additionally, this population displays characteristic morpho-physiological features in the juvenile and adult hippocampus. Therefore, the apparently homogeneous glutamatergic cell population likely displays a morpho-functional diversity rooted in temporal embryonic origins.

Pioneer glutamatergic cells develop into a morpho-functionally distinct population in the juvenile CA3 hippocampus.

Brain-machine interfaces (BMI) were born to control "actions from thoughts" in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI-a neuromorphic chip for brain repair-to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary "bottom-up" approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of "finite size networks" which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Les interfaces cerveau-machine (IMC) sont n&eacute;s au contr&ocirc;le &laquo; des actions de pens&eacute;es &raquo; afin de r&eacute;cup&eacute;rer le moteur capacit&eacute; des patients avec une d&eacute;ficience connectivit&eacute; fonctionnelle entre le syst&egrave;me nerveux central et p&eacute;riph&eacute;rique. L&#39;objectif final de nos travaux est l&#39;&eacute;laboration d&#39;une nouvelle puce de validation, un IMC neuromorphic pour cerveau-r&eacute;paration &agrave; reproduire l&#39;organisation fonctionnelle d&#39;une partie endommag&eacute;e du syst&egrave;me nerveux central. Pour atteindre cet objectif ambitieux, nous avons impl&eacute;ment&eacute; une approche multidisciplinaire de la &laquo; bottom-up &raquo; dans laquelle les r&eacute;seaux in vitro sont le paradigme pour le d&eacute;veloppement d&#39;un in silico mod&egrave;le &ecirc;tre int&eacute;gr&eacute;e &agrave; un dispositif neuromorphic. Dans cet article, nous pr&eacute;sentons la strat&eacute;gie globale et mettre l&#39;accent sur les diff&eacute;rents &eacute;l&eacute;ments constitutifs de nos &eacute;tudes: (i) l&#39;exp&eacute;rimental caract&eacute;risation et mod&eacute;lisation des &laquo; r&eacute;seaux de taille finie &raquo;, qui repr&eacute;sentent les circuits auto-organis&eacute;e plus petits et plus g&eacute;n&eacute;rales capables de g&eacute;n&eacute;rer une dynamique collective spontan&eacute;e ; (ii) l&#39;induction des l&eacute;sions dans les r&eacute;seaux de neurones et la pr&eacute;paration du cerveau entier avec une attention particuli&egrave;re sur l&#39;impact sur l&#39;organisation fonctionnelle des circuits ; (iii) la premi&egrave;re production d&#39;une puce neuromorphic capable d&#39;appliquer un mod&egrave;le en temps r&eacute;el des r&eacute;seaux neurones. Une caract&eacute;risation dynamique des circuits taille finie avec r&eacute;solution unicellulaire est fournie. Un mod&egrave;le de r&eacute;seau neuronal bas&eacute; sur neurones d&#39;izhikevich a &eacute;t&eacute; en mesure de reproduire les observations exp&eacute;rimentales. Changements dans la dynamique des circuits neuronaux induits par des l&eacute;sions isch&eacute;miques et optiques sont pr&eacute;sent&eacute;s respectivement pour les r&eacute;seaux de neurones in vitro et pour une pr&eacute;paration de tout le cerveau. Enfin, la mise en &oelig;uvre d&#39;une puce neuromorphic reproduisant la dynamique du r&eacute;seau en temps quasi r&eacute;el (pr&eacute;cision de 10 ns) est pr&eacute;sent&eacute;.

&Eacute;tudes in vitro &agrave; grande &eacute;chelle exp&eacute;rimentales et th&eacute;oriques pour la r&eacute;alisation de bi-directional cerveau-proth&egrave;ses.

Interfacce cervello-macchina (BMI) sono nati a controllo &quot;azioni da pensieri&quot; al fine di recuperare la motricit&agrave; dei pazienti con alterata connettivit&agrave; funzionale tra il sistema nervoso centrale e periferico. L&#39;obiettivo finale dei nostri studi &egrave; lo sviluppo di un nuovo chip di proof-of-concept, un BMI neuromorphic per riparazione di cervello-a riprodurre l&#39;organizzazione funzionale di una parte danneggiata del sistema nervoso centrale. Per raggiungere questo ambizioso obiettivo, abbiamo implementato un approccio &quot;bottom-up&quot; multidisciplinare in cui le reti in vitro sono il paradigma per lo sviluppo di un in silico modello ad essere incorporati in un dispositivo di neuromorphic. In questo libro ci presenta la strategia complessiva e concentrarsi sui diversi blocchi dei nostri studi: (i) la sperimentale caratterizzazione e modellazione delle &quot;reti di dimensione finita&quot;, che rappresentano i circuiti auto-organizzati pi&ugrave; piccoli e pi&ugrave; generali in grado di generare dinamiche collettive spontanee; (ii) l&#39;induzione di lesioni in reti neuronali e la preparazione del cervello intero con speciale attenzione all&#39;impatto sull&#39;organizzazione funzionale dei circuiti; (iii) la prima produzione di un chip di neuromorphic in grado di implementare un modello in tempo reale delle reti neuronali. Viene fornita una caratterizzazione dinamica dei circuiti di dimensione finita con risoluzione della singola cella. Un modello di rete neurale basato su neuroni Izhikevich era in grado di replicare alle osservazioni sperimentali. Cambiamenti nella dinamica dei circuiti neuronali indotti da lesioni ischemiche e ottiche sono presentati rispettivamente per le reti neuronali in vitro e per una preparazione intero cervello. Infine l&#39;implementazione di un chip neuromorphic riproducendo le dinamiche di rete in tempo quasi-real (precisione 10 ns) &egrave; presentato.

Studi in vitro su larga scala teorici e sperimentali per la realizzazione del cervello-protesi bi-direzionale.

Linking the structural connectivity of brain circuits to their cooperative dynamics and emergent functions is a central aim of neuroscience research. Graph theory has recently been applied to study the structure-function relationship of networks, where dynamical similarity of different nodes has been turned into a "static" functional connection. However, the capability of the brain to adapt, learn and process external stimuli requires a constant dynamical functional rewiring between circuitries and cell assemblies. Hence, we must capture the changes of network functional connectivity over time. Multi-electrode array data present a unique challenge within this framework. We study the dynamics of gamma oscillations in acute slices of the somatosensory cortex from juvenile mice recorded by planar multi-electrode arrays. Bursts of gamma oscillatory activity lasting a few hundred milliseconds could be initiated only by brief trains of electrical stimulations applied at the deepest cortical layers and simultaneously delivered at multiple locations. Local field potentials were used to study the spatio-temporal properties and the instantaneous synchronization profile of the gamma oscillatory activity, combined with current source density (CSD) analysis. Pair-wise differences in the oscillation phase were used to determine the presence of instantaneous synchronization between the different sites of the circuitry during the oscillatory period. Despite variation in the duration of the oscillatory response over successive trials, they showed a constant average power, suggesting that the rate of expenditure of energy during the gamma bursts is consistent across repeated stimulations. Within each gamma burst, the functional connectivity map reflected the columnar organization of the neocortex. Over successive trials, an apparently random rearrangement of the functional connectivity was observed, with a more stable columnar than horizontal organization. This work reveals new features of evoked gamma oscillations in developing cortex.

Quantifying network properties in multi-electrode recordings: spatiotemporal characterization and inter-trial variation of evoked gamma oscillations in mouse somatosensory cortex in vitro.

It has recently been discovered that single neuron stimulation can impact network dynamics in immature and adult neuronal circuits. Here we report a novel mechanism which can explain in neuronal circuits, at an early stage of development, the peculiar role played by a few specific neurons in promoting/arresting the population activity. For this purpose, we consider a standard neuronal network model, with short-term synaptic plasticity, whose population activity is characterized by bursting behavior. The addition of developmentally inspired constraints and correlations in the distribution of the neuronal connectivities and excitabilities leads to the emergence of functional hub neurons, whose stimulation/deletion is critical for the network activity. Functional hubs form a clique, where a precise sequential activation of the neurons is essential to ignite collective events without any need for a specific topological architecture. Unsupervised time-lagged firings of supra-threshold cells, in connection with coordinated entrainments of near-threshold neurons, are the key ingredients to orchestrate population activity.

Article	Year
Toward the neurocomputer: image processing and pattern recognition with neuronal cultures. IEEE transactions on bio-medical engineering\| PubMed ID: 15759567	2005
Statistical properties of information processing in neuronal networks. The European journal of neuroscience\| PubMed ID: 16324130	2005
Embryonic stem cell-derived neurons form functional networks in vitro. Stem cells (Dayton, Ohio)\| PubMed ID: 17110621	2007
Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence. Analytical and bioanalytical chemistry\| PubMed ID: 17203251	2007
On the dynamics of the spontaneous activity in neuronal networks. PloS one\| PubMed ID: 17502919	2007
Sequential generation of two distinct synapse-driven network patterns in developing neocortex. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 19036979	2008
Dissecting functional connectivity of neuronal microcircuits: experimental and theoretical insights. Trends in neurosciences\| PubMed ID: 21459463	2011
Pioneer GABA cells comprise a subpopulation of hub neurons in the developing hippocampus. Neuron\| PubMed ID: 21867885	2011
The role of the neuro-astro-vascular unit in the etiology of ataxia telangiectasia. Frontiers in pharmacology\| PubMed ID: 23060792	2012
Pioneer glutamatergic cells develop into a morpho-functionally distinct population in the juvenile CA3 hippocampus. Nature communications\| PubMed ID: 23271650	2012
In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses. Frontiers in neural circuits\| PubMed ID: 23503997	2013
Quantifying network properties in multi-electrode recordings: spatiotemporal characterization and inter-trial variation of evoked gamma oscillations in mouse somatosensory cortex in vitro. Frontiers in computational neuroscience\| PubMed ID: 24137127	2013
Clique of functional hubs orchestrates population bursts in developmentally regulated neural networks. PLoS computational biology\| PubMed ID: 25255443	2014

Paolo Bonifazi

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