Developing neural crest cells in culture: correlation of cytochrome oxidase activity with SSEA-1 and dopamine-β-hydroxylase immunoreactivity

Abstract

This study examines possible changes in energy demands by developing neural crest cells in vitro using cytochrome oxidase (C.O.) histochemistry and immunohistochemial labeling of adrenergic (automatic) cells and primary sensory neurons. Cytochrome oxidase is a key enzyme for oxidative metabolism and energy production, and it is used as a sensitive metabolic marker for neurons in the brain and dorsal root ganglia. In primary neural crest cell cultures, C.O. staining intensities differ among 4 distinct cellular populations (sensory neurons, adrenergic cells, pigment cells, and non-neuronal crest cells). At all stages, pigment cells exhibit extremely low C.O. staining. Neurons (both primary sensory and adrenergic cells) have higher C.O. activity than other cell types such as non-neuronal crest cells. This indicates that neurons have higher energy demands and presumably higher levels of functional activity than other types at least under the present culture conditions. All neurons in neural crest cell cultures elevate their demands during development, implying that energy metabolism and functional activity increase with neuronal maturation. At all stages, early determined sensory neurons exhibit more intense C.O. staining than late-developing sensory neurons. The difference in C.O. activity between the two population of sensory neurons may be caused by different levels of functional activity due to their different time course of development. Tetrodotoxin (TTX) at 0.5–1 μM concentrations causes a decrease in the level of C.O. activity in the early determined sensory neurons, which may be correlated with a decrease in the functional activity of these neurons. Triple staining combining C.O. histochemistry with direct immunofluorescence of antibodies against the stage specific embryonic antigen-1 (SSEA-1, which labels quail sensory neurons) and dopamine-β-hydroxylase (DBH, which labels adrenergic cells) distinguish the levels of C.O. activity between sensory neurons and autonomic cells DBH + cells exhibit relatively low C.O. staining. However, the C.O. activity among SSEA-1 + neurons varies from high to low levels. In general, SSEA-1 + sensory neurons are much more C.O. reactive than DBH + autonomic cells. This suggest that developing sensory neurons in culture may have higher spontaneous and/or synaptic activity than autonomic neurons.