The acquisition and expression of a peptidergic phenotype in the grasshopper embryo

We have examined the developmental acquisition and regional expression of the neurotransmitter proctolin in neurons of the grasshopper embryo, using both assay and immunocytochemistry. There are four stages in the acquisition and accumulation of proctolin in the embryo. The first stage (50 to 60% embryogenesis) consists of a slow accumulation of proctolin prior to the onset of central nervous system (CNS) staining. During the second stage proctolin levels increase at a 5-fold faster rate. During this stage, which ends at 70% of development, a stable pattern of staining is established in the ganglia. The third stage (70 to 95% embryogenesis) consists of a rapid accumulation of peripheral proctolin levels that occurs principally in motor endings, without a significant increase in the CNS. This stage also correlates with the onset of peripheral staining. During the fourth stage, over the final 5% of embryogenesis, there is a 20% loss of proctolin in the periphery. We have attempted to relate these changes to developmental events occurring in identified clusters of neurons, by examining the development of neuromuscular connections made by embryonic terminal ganglion motoneurons to the intrinsic muscles of the hindgut. Of the 30 hindgut motoneurons, only six anterior medial (AM) efferents are proctolin immunoreactive in the embryo. Their axons establish a dense proctolin-staining arborization over the intrinsic muscles. The peptide accumulates in the endings as staining appears in the terminal ganglion. Hindgut proctolin levels increase substantially and then decline in a manner similar to that found for total peripheral proctolin. By the end of embryogenesis the endings will release proctolin, in a calcium-dependent manner, when depolarized. The six proctolin-staining AM neurons constitute a simple model system for studying the determination and expression of neurotransmitters in identified embryonic cells.