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

Abstract

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, 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 mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.

Vesicle capture, not delivery, scales up neuropeptide storage in neuroendocrine terminals

“…Muscle 6/7 synapse type Ib boutons were studied in Figs. 1-3 with elav-GAL4 UAS-Anf-GFP larvae (19). Type III boutons were studied in upstream activating sequence (UAS)-Anf-GFP; ccap-GAL4 larvae.…”

“…driven by a panneuronal driver shows greater accumulation of neuropeptide in Drosophila type III boutons on muscle 12 than in type Ib boutons of the muscle 6/7 synapse (19). Panneuronal expression is not convenient for studying type III boutons, however, because of the presence of closely apposed type Ib, Is, and II boutons, which arise from different neurons.…”

Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis

“…Indeed, when we blocked neuropeptide processing through Amon function, or neuropeptide release by reducing Caps, which is required for dense core vesicle release 58 , we could fully suppress the tll knockdown-induced aggression response. Finally, we used a heterologous neuropeptide GFP reporter 59 to visualize the effect of tll knockdown on neuropeptide release from PI neurons and showed that the reporter nearly completely vanishes from PI neurons when tll is knocked down. The identity of the specific neuropeptide(s) that regulate aggression through this transcriptional module is currently not known.…”

Energy-Dependent Modulation of Glucagon-Like Signaling inDrosophilavia the AMP-Activated Protein Kinase

“…The UAS-AMPK transgenes were reported previously : UAS-snf1A K57A [Bloomington Stock (BL) # 32112], UAS-snf1A (BL# 32108), UAS-snf1A-RNAi (BL# 32371), and UAS-snf4-RNAi (BL# 34726). Other stocks used in this study were the AKH-GAL4 (BL # 25683) (Lee and Park 2004), UAS-TeTX (BL# 28839) (Sweeney et al 1995), UAS-AKH-RNAi (BL#34960), UAS-rpr (BL# 5823), GAL80 (BL#7018) (McGuire et al 2003), UAS-GCaMP (BL# 32236), and UAS-ANF-GFP (BL# 7001) (Rao et al 2001), all procured from the Bloomington Stock Center (Table 1). The AKHR 01 and AKHR rev lines were kind gifts from Ronald Kuhnlein (Gronke et al 2007).…”