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

The roles of protein kinase C and the MAP-kinase extracellular receptor kinase in structural changes associated with long-term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N-methyl-d-aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis-dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12-myristate 13-acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12-myristate 13-acetate. Both the conditioning medium and phorbol 12-myristate 13-acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long-term increase in network activity and functional synaptic connectivity among the cultured neurons.

Protein kinase C and ERK involvement in dendritic spine plasticity in cultured rodent hippocampal neurons.

GABAergic interneurons of the hippocampus play an important role in the generation of behaviorally relevant network oscillations. Among this heterogeneous neuronal population, somatostatin (SOM)-positive oriens-lacunosum moleculare (O-LM) interneurons are remarkable because they are tuned to operate at theta frequencies (6-10 Hz) in vitro and in vivo. Recent studies show that a high proportion of glutamatergic synapses that impinge on O-LM interneurons are mediated by kainate receptors (KA-Rs). In the present study, we thus tested the hypothesis that KA-Rs transmit afferent inputs in O-LM neurons during synaptic stimulation at theta frequency. We combined multibeam two-photon calcium imaging in hippocampal slices from SOM-enhanced green fluorescent protein (EGFP) mice, to record the activity of SOM cells as well as hundreds of neurons simultaneously, and targeted electrophysiological recordings and morphological analysis to describe the morphofunctional features of particular cells. We report that EGFP-positive O-LM neurons are the only subtype of interneuron that reliably follows synaptic stimulation of the alveus in the theta frequency range. Electrophysiological recordings revealed the crucial contribution of KA-Rs to the firing activity and to the glutamatergic response to theta stimuli in O-LM cells compared with other cell types. The reliable activation of O-LM cells in the theta frequency range did not simply result from the longer kinetics of KA-R-mediated postsynaptic events (EPSP(KA)) but presumably from a specific interaction between EPSP(KA) and their intrinsic active membrane properties. Such preferential processing of excitatory inputs via KA-Rs by distally projecting GABAergic microcircuits could provide a key role in theta band frequency oscillations.

Synaptic kainate receptors tune oriens-lacunosum moleculare interneurons to operate at theta frequency.

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity.

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.

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.

The effect of Alzheimer's disease pathology on activity of individual neocortical neurons in the intact neural network remains obscure. Ongoing spontaneous activity, which constitutes most of neocortical activity, is the background template on which further evoked-activity is superimposed. We compared in vivo intracellular recordings and local field potentials (LFP) of ongoing activity in the barrel cortex of APP/PS1 transgenic mice and age-matched littermate Controls, following significant amyloid-β (Aβ) accumulation and aggregation. We found that membrane potential dynamics of neurons in Aβ-burdened cortex significantly differed from those of nontransgenic Controls: durations of the depolarized state were considerably shorter, and transitions to that state frequently failed. The spiking properties of APP/PS1 neurons showed alterations from those of Controls: both firing patterns and spike shape were changed in the APP/PS1 group. At the population level, LFP recordings indicated reduced coherence within neuronal assemblies of APP/PS1 mice. In addition to the physiological effects, we show that morphology of neurites within the barrel cortex of the APP/PS1 model is altered compared to Controls. These results are consistent with a process where the effect of Aβ on spontaneous activity of individual neurons amplifies into a network effect, reducing network integrity and leading to a wide cortical dysfunction.

Article	Year
Protein kinase C and ERK involvement in dendritic spine plasticity in cultured rodent hippocampal neurons. The European journal of neuroscience\| PubMed ID: 12823460	2003
Synaptic kainate receptors tune oriens-lacunosum moleculare interneurons to operate at theta frequency. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17804617	2007
Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity. Developmental neurobiology\| PubMed ID: 18327766	2008
The role of the neuro-astro-vascular unit in the etiology of ataxia telangiectasia. Frontiers in pharmacology\| PubMed ID: 23060792	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
Amyloid-β disrupts ongoing spontaneous activity in sensory cortex. Brain structure & function\| PubMed ID: 25523106	2014

Miri Goldin

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