Pronounced cellular diversity and extrasynaptic location of nicotinic acetylcholine receptor subunit immunoreactivities in the chicken pretectum

Abstract

The diversity of nicotinic ACh receptor (AChR) expression in the chick lateral spiriform nucleus (SpL) was assessed using subunit-specific monoclonal antibodies (mAbs) and laser scanning confocal microscopy. The late embryonic SpL was immunoreactive for mAbs against the α2, α5, α7, α8, and β2 AChR subunits. Distinct neuronal cell classes were determined using pair- wise staining of mAbs. Approximately 90% of the neurons in the SpL contained both α5-like immunoreactivity (Ll) and β2-Ll, with no neurons having only one of these subunit-Lls. Approximately 70% of the neurons contained α2-Ll. All α2-Ll neurons contained α5/β2-Ll; thus, neurons having α2-Ll are a subset of those having α5- and β2-Ll. Fewer neurons, approximately 20%, contained α7-Ll. A subset of α7-positive neurons were immunoreactive for other subunits; for example, some α7-positive neurons also contained α2- Ll. Fewer than 15% of the neurons contained α8-Ll. Some of the α8-Ll- containing neurons contained α7-Ll. The 14 week post-hatch SpL resembles the late embryonic nucleus in the percentage of neurons immunoreactive for α2, α5, α7, αβ, and β2 AChR subunits, and in the presence of multiple classes based on AChR subunit immunoreactivity. In addition, α4-Ll was found in about 20% of the 14 week SpL neurons. Double-label immunofluorescence experiments with mAbs to AChRs and to synaptic vesicle antigens showed that most clusters of α5-Ll and β2-Ll are extrasynaptic. The pronounced diversity of AChR subunit expression and the extrasynaptic location of AChR- Ll suggest that AChR-like molecules in the SpL do not function solely to respond to transmitter focally released from presynaptic terminals.