In-mold patterning and actionable axo-somatic compartmentalization for on-chip neuron culture.

Oriented neuronal networks with controlled connectivity are required for many applications ranging from studies of neurodegeneration to neuronal computation. To build such networks in vitro, an efficient, directed and long lasting guidance of axons toward their target is a pre-requisite. The best guidance achieved so far, however, relies on confining axons in enclosed microchannels, making them poorly accessible for further investigation. Here we describe a method providing accessible and highly regular arrays of axons, emanating from somas positioned in distinct compartments. This method combines the use of a novel removable partition, allowing soma positioning outside of the axon guidance patterns, and in-mold patterning (iMP), a hybrid method combining chemical and mechanical cell positioning clues applied here for the first time to neurons. The axon guidance efficiency of iMP is compared to that of conventional patterning methods, e.g. micro-contact printing (chemical constraints by a poly-l-lysine motif) and micro-grooves (physical constraints by homogeneously coated microstructures), using guiding tracks of different widths and spacing. We show that iMP provides a gain of 10 to 100 in axon confinement efficiency on the tracks, yielding mm-long, highly regular, and fully accessible on-chip axon arrays. iMP also allows well-defined axon guidance from small populations of several neurons confined at predefined positions in µm-sized wells. iMP will thus open new routes for the construction of complex and accurately controlled neuronal networks.

Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network.

INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level. RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (µFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied ß-amyloid (Aß) peptides locally to the different cellular compartments. We observed that Aß application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aß deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aß peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aß stress at the network level we developed new µFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aß application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration. CONCLUSION: Thanks to µFD-based reconstructed neuronal networks we evaluated the distant effects of local Aß stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aß production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aß can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

A review of microfabrication and hydrogel engineering for micro-organs on chips.

This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or "organs on chips". First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.

Synapto-protective drugs evaluation in reconstructed neuronal network.

Chronic neurodegenerative syndromes such as Alzheimer's and Parkinson's diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile "brain on chip" platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD⁺ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers.

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.

Copper transfer from Cu-Abeta to human serum albumin inhibits aggregation, radical production and reduces Abeta toxicity.

Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Abeta and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Abeta aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper-exchange reaction between Abeta and HSA or the tetrapeptide DAHK (N-terminal Cu-binding domain of HSA) and the consequence of this exchange on Abeta-induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu(II) from Abeta rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu-induced aggregation of Abeta, 3) HSA and DAHK suppressed the catalytic HO(.) production in vitro and ROS production in neuroblastoma cells generated by Cu-Abeta and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu-Abeta with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu(II)-HSA than Cu-DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu(II)-Abeta interaction. The repercussion of the competition between HSA and Abeta to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.