Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster

Neuropeptides affect an extremely diverse set of physiological processes. N europeptides are often coreleased with neurotransmitters but, unlike neurot ransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropep tide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, trans port, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos . By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushr oom bodies, antennal lobe, and the central complex. At the larval neuromusc ular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain a lmost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approx imately 60% of the tagged neuropeptide was released from NMJs of both muscl es in 15 min, as assayed by decreased fluorescence. Although the elav promo ter was equally active in the motor neurons that innervate both NMJs 6/7 an d 12, NMJ 12 contained 46-fold more neuropeptide and released much more pro ANF-EMD during stimulation than did NMJ 6/7. Our results suggest that pepti dergic neurons have an enhanced ability to accumulate and/or release neurop eptides as compared to neurons that primarily release classical neurotransm itters. (C) 2001 John Wiley & Sons, Inc.