Chromaffin cell heterogeneity of process formation and neuropeptide content under control and nerve growth factor-altered conditions in cultures of chick embryonic adrenal gland

Abstract

Adrenal glands from embryonic day 11 (E-11) chicks were cryostat-sectioned, and it was determined that tyrosine hydroxylase-like immunoreactive (TLI) cells, somatostatin-like immunoreactive (SLI) cells, and methionine-enkephalin-like immunoreactive (ELI) cells occupied chromaffin regions of the gland. Similar age adrenals were dissociated, and the cells were cultured under serum-free conditions. Cultured TLI cells, ELI cells, and SLI cells were characterized according to cell size, cell number, and neurite formation. ELI and SLI cells composed two largely separate populations, with SLI cells tending to have larger cell areas, to be more numerous, and to be less likely to form neurites than ELI cells. The population of TLI cells, although unique in itself, was diverse and numerous enough to include all or portions of the neuropeptide-immunoreactive populations. Neurites of some cells from each of the above populations were strongly immunoreactive for alpha neurofilament protein, and for NAPA73 neurofilament-associated protein. However, neurites could also be observed in all populations that showed poor immunoreactivity for these cytoskeletal proteins. Exogenously added NGF significantly increased neurite-like process formation among TLI and ELI cells, but not among SLI cells. Reductions in the number of neurite-like processes following treatment with anti-nerve growth factor (NGF) were not significant for any of the populations. However, if shorter and broader process were included, anti-NGF caused a significant reduction in total cell processes among TLI and ELI cells. Anti-NGF inhibition of process formation among ELI cells could be reversed with exogenous NGF. Neither NGF or anti-NGF treatments showed a significant effect on cell numbers among TLI and ELI populations. The implications are that a compound of antigenic and physiological similarity to mouse salivary NGF is made by embryonic chick adrenal cells in culture, but the effects of NGF do not appear to be the same for all neural-crest-derived cells from the adrenal, and greater heterogeneity of phenotypes may exist among chromaffin cells than has previously been accepted. Some questions are also raised concerning the neurite-like nature of processes formed by some chromaffin cells in vitro.