Nano-volume drop patterning for rapid on-chip neuronal connect-ability assays

Abstract

The ability of neurons to extend projections and to form physical connections among them (i.e., "connect-ability") is altered in several neuropathologies. The quantification of these alterations is an important read-out to investigate pathogenic mechanisms and for research and development of neuropharmacological therapies, however current morphological analysis methods are very time-intensive. Here, we present and characterize a novel on-chip approach that we propose as a rapid assay. Our approach is based on the definition on a neuronal cell culture substrate of discrete patterns of adhesion protein spots (poly-d-lysine, 23 ± 5 μm in diameter) characterized by controlled inter-spot separations of increasing distance (from 40 μm to 100 μm), locally adsorbed in an adhesion-repulsive agarose layer. Under these conditions, the connect-ability of wild type primary neurons from rodents is shown to be strictly dependent on the inter-spot distance, and can be rapidly documented by simple optical read-outs. Moreover, we applied our approach to identify connect-ability defects in neurons from a mouse model of 22q11.2 deletion syndrome/DiGeorge syndrome, by comparative trials with wild type preparations. The presented results demonstrate the sensitivity and reliability of this novel on-chip-based connect-ability approach and validate the use of this method for the rapid assessment of neuronal connect-ability defects in neuropathologies.

Emergence of Bursting Activity in Connected Neuronal Sub-Populations

“…In the last decades, many studies have been presented, using various methods including surface modification by silane chemistry [9], photolithographic techniques [10], deep-UV lithography [11], soft lithography [12] and spot-arrays of adhesion molecules [13], [14]. Other methods were more recently used to organize networks by imposing physical constraints [15], [16].…”

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

“…This strategy, that required a careful optimization of the coating, assembly and sterilization procedures and the creation of a custom microalignment system, gave us some major advantages with respect to previously reported works (e.g., the long-term survival of our cells, i.e., up to 8 weeks in vitro). Specifically, it provided us with a more stable and reliable long-term confinement 24 with respect to strategies based on chemical patterning [23][24][25][26][27][28] .…”

Brain cells and neuronal networks: Encounters with controlled microenvironments

“…This study enlightened the differences in the exploration range of growth cones between different neuron types (12 lm in chick embryo brain neurons, 50 lm for mouse neonatal DRG neurons), setting the minimal distance between branches of a neuronal network. Then, more complex micropatterns built from stripes interconnected through adhesive spots dedicated to soma adhesion have been used to produce in vitro organized neuronal networks [114,115]. Patch-clamps recordings have been performed on mature neuronal networks grown on such patterned surfaces (Fig.…”