Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Abstract

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here. Video LinkThe video component of this article can be found at

Intracellular G-actin targeting of peripheral sensory neurons by the multifunctional engineered protein C2C confers relief from inflammatory pain

“…To confirm the occurrence of similar activity by C2C, its effect on actin polymerization was tested. Primary chicken sensory neurons were cultured as described previously 25 , followed by treatment with 60 nM C2C or latrunculin A and staining of treated cells with the F-actin stain phalloidin 26 . Microscopic analysis showed a reduction in F-actin staining for both C2C and latrunculin A treated cells compared to an untreated control cell line though the effect of latrunculin A appeared more significant at this dose (Fig.…”

“…Because depolymerization of F-actin mediated by latrunculin A has been shown to disrupt calcium influx 30 , the ability of C2C to inhibit calcium influx was examined. Primary chicken sensory cells prepared as described 25 were treated with C2C and then with the calcium fluorescent dye, Fluo-4 31 . C2C treatment reduced calcium influx as indicated by a reduction in fluorescence at a range of concentrations of C2C (Fig.…”

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth

“…Embryonic day 12 eggs were opened in a biological safety cabinet to collect the E12 embryo, which was cleaned, and organs were removed (in compliance with UK Home Office Animals [Scientific Procedures] Act 1986 and definition of chicken as a Protected Animal from E14). DRGs were dissected and under a dissecting microscope, the remains of the root were cut (Powell, Vinod, & Lemons, 2014). DRGs were seeded at a density of one per well on TCP and on bioactive surfaces, with Dulbecco's modified Eagle's medium (DMEM; Sigma) plus 10% (v/v) fetal bovine serum (PAN Biotech, Germany), 1% (w/v) l ‐glutamine (Sigma), 1% (w/v) penicillin/streptomycin (P/S; Sigma), 0.25% (w/v) amphotericin B (Sigma), and NGF, BDNF, or NGF plus BDNF at the above concentrations.…”

Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia

“…Dorsal root ganglia (DRGs) were dissected from stage 33 (�E8) chick embryos using sterile fine tweezers according to a protocol adapted from Powell et al, 2014 [27]. The Wake Forest Institutional Animal Care and Use Committee (WF IACUC) provided us a waiver from ethics board review.…”