Selective expression of high-affinity nerve growth factor receptors on tyrosine hydroxylase-containing neuron-like cells in neural crest cultures

Abstract

A subpopulation of cultured neural crest cells undergoing differentiation have receptors for nerve growth factor (NGF) that exhibit a binding constant similar to that of the low-affinity NGF binding site (3.2 nM). Recent studies have shown that NGF receptors are not present on neuron-like cells immunoreactive for tyrosine hydroxylase (TH), serotonin, or vasoactive intestinal polypeptide. Since tissues innervated by sympathetic neurons in vivo produce NGF, we sought to determine whether NGF deficits in the tissue culture microenvironment may be one parameter preventing the expression of NGF receptors on TH-containing neuron-like cells. Neural crest cultures were therefore grown in complete tissue culture medium (15% fetal bovine serum and 5% chicken embryo extract), with or without exogenous NGF (50 ng/ml). Examination of light-microscopic radioautographs following incubation with 125I-NGF revealed that, if the cultures were supplemented with NGF for 7 d, approximately 33% of neuron-like cells exhibiting TH-like immunoreactivity possessed NGF receptors. There were no obvious morphological differences between TH-containing cells that did or did not have NGF receptors. Scatchard analysis of cultures grown under these conditions again demonstrated the sole presence of the low-affinity form of the NGF receptor (K(d), 3.4 nM). Embryonic catecholaminergic sympathetic neurons exhibit both high- and low-affinity forms of the NGF receptor, raising the possibility that the Scatchard analysis may not have been sensitive enough to detect the high-affinity form of the receptor on a relatively small population of cells. Therefore we used a morphological approach that took advantage of the different dissociation rates of the 2 receptor types. Data obtained from such a technique indicated that TH-containing neuron-like cells in neural crest cultures grown in the continuous presence of NGF have high-affinity NGF receptors; TH-like immunoreactive cells with silver grains were still detected following treatment that eliminated 125I-NGF binding to low-affinity sites. It remains to be determined whether the effect of NGF on the appearance of NGF receptors on TH-containing neuron-like cells is due to survival of a particular subpopulation of cells or to up-regulation of the NGF receptor.