The ability to engineer living networks of interconnected neurons with specified connectivity would facilitate the study of synaptogenesis and information processing in the nervous system. Previously, we found that a neurite can be elicited from embryonic chick forebrain neurons by direct mechanical means using magnetic bead force application (MBFA); however, our previous studies and others focused on young, synapse-incompetent neurons. To address this issue, we tested cultures of embryonic chick forebrain neurons of varying age and found that neurites could be micromechanically elicited via MBFA at all ages tested, which ranged between 7 and 22 embryonic equivalent (EE) days (days in ovo plus days in vitro). The probability of neurite initiation was at least 40% for all ages, with a maximum of ∼80% after 2-4 days in vitro, and a decrease to ∼60% by day 10 in vitro. The force required to elicit a neurite was ∼1500 pN with a minimum of ∼700 pN at embryonic equivalent day 14. The probability of success was similar for two rates of force application (10 and 500 pN/s). Neurite initiation via micromechanical force is robust with respect to cell age, and micromechanical force can induce neurites in synapse-competent neurons.
Bibliographical noteFunding Information:
This study was supported by the NSF Biological Information Technology & Systems program (grant EIA-0130875) and the University of Minnesota Biomedical Engineering Institute. Thanks to Joseph Fass for helpful discussions on neuron culture and MBFA protocol. Thanks to Sanford Weisberg for help with statistical analysis and as a personal source for the R code to perform the delta method.
- Cell growth
- Neural network engineering
- β-1 Integrin