Nano-volume drop patterning for rapid on-chip neuronal connect-ability assays.
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.
Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy. silvia.dante@iit.it.
Alessia
A
Petrelli
Emanuele
E
Marconi
Marco
M
Salerno
Davide
D
De Pietri Tonelli
Luca
L
Berdondini
Silvia
S
Dante
D000818:Animals; D002478:Cells, Cultured; D004062:DiGeorge Syndrome / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D004622:Embryo, Mammalian / Q000166:cytology; D051379:Mice; D046210:Microfluidic Analytical Techniques; D018625:Microscopy, Atomic Force; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D011107:Polylysine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D059008:Time-Lapse Imaging
D011506:Proteins; D011107:Polylysine