Expression of Nicotinic Acetylcholine Receptors and Subunit mRNA Transcripts in Cultures of Neural Crest Cells

AbstractThe expression of neuronal phenotypic characteristics by neural crest-derived neurons is dependent upon both environmental cues and intrinsic properties. Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed by most neural crest-derived peripheral neurons; nevertheless, the factors that influence and modulate the early expression of nAChRs on neuronal precursor cells are unknown. We used RT-PCR to analyze the temporal sequence of the appearance of transcripts encoding α3, α5, α7, β2, and β4 nAChR subunits in cultures of quail neural crest cells. At 2 days in culture, mRNA encoding each of these nAChR subunits was detectable and, except for α7, remained so through 12 days. The appearance of NAPA 73 immunoreactivity during neutral crest cell migration suggested that some cells express neuronal characteristics early in development and that the expression of nAChR subunits may be cell autonomous for some neural crest-derived cells. Between 8 and 10 days in culture, a 2.0-kb transcript encoding α3 subunit appears in addition to the expected 3.5-kb transcript that is present at 8 days. nAChR protein was detected immunocytochemically at 7 days and was observed in immunoblots by 9 days. Wholecell patch clamp recordings demonstrated that functional nAChRs develop concurrently in a subset of neural crest-derived neurons. In addition, both neuronal and nonneuronal cells showed inward and delayed outward currents in response to step depolarization. The appearance of functional nAChRs on neural crest-derived cells, in vitro, occurs independently of innervation and of the components in growth medium. The data suggest that neural crest-derived cells express transcripts encoding nAChR subunits, possibly as an intrinsic property of some peripheral neurons, early in development