Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture

Abstract

Survival and development of chick ciliary ganglion neurons in vivo appear to depend on information from the embryonic eye structure that contains the postsynaptic targets of the neurons. We have tested embryonic eye extracts on ciliary ganglion neurons in dissociated cell culture for stimulation of growth and development. Control conditions were chosen that permitted the long term maintenance of the neurons in the absence of tissue extracts of conditioned medium. The conditions included coating the culture substratum with fibroblast material and increasing the K+ concentration in the culture medium to 25 mM. Neurons survived for at least 3 weeks in control conditions. Two major components were resolved in eye extracts that stimulated growth and development of the neurons above the basal levels obtained with control conditions. One component, with an apparent molecular weight of about 2 X 10(4) by gel filtration analysis, stimulated neuronal growth without increasing the levels of choline acetyltransferase activity per neuron. The second component, with an apparent molecular weight of about 5 X 10(4), increased development of choline acetyltransferase levels per neuron but had no effect on neuronal growth. Both components were effective in normal K+ as well as 25 mM K+. These components may represent mechanisms by which the postsynaptic target tissue acts in vivo to direct the growth and development of ciliary ganglion neurons.

Rapid Activity-Driven SNARE-Dependent Trafficking of Nicotinic Receptors on Somatic Spines

“…Dissociated embryonic day 8 (E8) CG neurons were grown in culture for 6 -8 d on glass coverslips coated with poly-D-lysine, fibronectin, and lysed fibroblasts as described previously (Nishi and Berg, 1981;Zhang et al, 1994). Dissociated E14 CG neurons were prepared and maintained for 1-4 h on glass coverslips lacking the lysed fibroblasts.…”

Patch‐clamp analysis of glycine‐induced currents in chick ciliary ganglion neurons.

“…Briefly, the ganglia were taken from embryos, killed by rapid decapitation, cut in half, incubated with 1 mg ml-' collagenase (Type A, Boehringer Mannheim) for 30 min at 37°C, and transferred to culture medium consisting of Eagle's minimal essential medium (GIBCO) supplemented with 3% (v/v) chick embryo eye extract (Nishi & Berg, 1981). The cells were dispersed by trituration with fire-polished pasteur pipettes and plated on a substratum of poly-D-lysine in 35 mm Costar culture dishes.…”

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

“…Dissociated ciliary ganglion cell cultures were prepared from 8 d chick embryos and grown at 37°C in an atmosphere of 5% CO,/95% air on a substratum of collagen and lysed fibroblasts as previously described (Nishi and Berg,198 1). Cultures contained about 1.4 x lo4 neurons/l6 mm well (or 7 x lo4 neurons/35 mm dish for sucrose gradient experiments) and were grown in a culture medium consisting of Eagle's minimal essential medium containing 10% (vol/ vol) heat-inactivated horse serum, 50 units/ml penicillin, 50 FLg/ml streptomycin, and 3% (vol/vol) embryonic eye extract (Nishi and Berg, 1981). Culture medium was replaced at 2-3 d intervals and cultures were taken for experiments at 4 d unless otherwise indicated.…”

“…White leghorn chick embryos were obtained locally and maintained at 39°C in a humidified incubator. Tissue extracts and culture media were prepared as previously described (Nishi and Berg, 1981). Culture media components were obtained from Grand Island Biological Co. except for horse serum, which was obtained from Whitakker M.A.…”