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

Abstract

Neurons vary in their capacity to produce, store, and release neuropeptides packaged in dense-core vesicles (DCVs). Specifically, neurons used for cotransmission have terminals that contain few DCVs and many small synaptic vesicles, whereas neuroendocrine neuron terminals contain many DCVs. Although the mechanistic basis for presynaptic variation is unknown, past research demonstrated transcriptional control of neuropeptide synthesis suggesting that supply from the soma limits presynaptic neuropeptide accumulation. Here neuropeptide release is shown to scale with presynaptic neuropeptide stores in identified Drosophila cotransmitting and neuroendocrine terminals. However, the dramatic difference in DCV number in these terminals occurs with similar anterograde axonal transport and DCV half-lives. Thus, differences in presynaptic neuropeptide stores are not explained by DCV delivery from the soma or turnover. Instead, greater neuropeptide accumulation in neuroendocrine terminals is promoted by dramatically more efficient presynaptic DCV capture. Greater capture comes with tradeoffs, however, as fewer uncaptured DCVs are available to populate distal boutons and replenish neuropeptide stores following release. Finally, expression of the Dimmed transcription factor in cotransmitting neurons increases presynaptic DCV capture. Therefore, DCV capture in the terminal is genetically controlled and determines neuron-specific variation in peptidergic function.nerve terminal | secretory granule | neurotransmission | LDCV

Limited distal organelles and synaptic function in extensive monoaminergic innervation

“…For neuropeptide release, the impact of fewer DCVs is apparent from previous experiments, which show that release scales with presynaptic neuropeptide stores (e.g. Bulgari et al, 2014). Ultrastructural studies confirm that rapid stimulation- induced Ca 2+ -dependent FM dye uptake into Drosophila boutons is mediated by endocytosis to form SSVs (e.g.…”

“…To test this hypothesis, we used approaches that previously revealed vesicle circulation in type Ib boutons. First, we examined the age of DCVs in proximal and distal type II boutons by imaging a timer construct (ANF tagged with monomeric Kusabira Green Orange, mK-GO, driven by 386Y-Gal4) that converts from green to red fluorescence over hours (Tsuboi et al, 2010;Bulgari et al, 2014). Fluorescence red to green ratio (F red/green ) showed that, in contrast to findings in type Ib boutons that are supplied by vesicle circulation, DCVs in distal type II boutons are older than their proximal cohort (Fig.…”

“…This process has been termed capture and has been measured as the decrease in DCV flux entering and leaving a bouton (Shakiryanova et al, 2006;Wong et al, 2012;Bulgari et al, 2014Bulgari et al, , 2017Cavolo et al, 2016). Notably, this parameter encompasses capture initiation, which when measured over minutes does not quantify the much longer duration of capture (Shakiryanova et al, 2006).…”

“…Recently, it was shown that glutamatergic type Ib en passant boutons at the Drosophila neuromuscular junction (NMJ) are supplied equivalently with functional DCVs by vesicle circulation and bidirectional capture (Wong et al, 2012(Wong et al, , 2015. Furthermore, synaptic capture of DCVs increases following activity and is constitutively elevated by a transcription factor in specialized neuroendocrine neurons (Shakiryanova et al, 2006;Bulgari et al, 2014;Cavolo et al, 2016). Disease-related proteins such as the fragile-X syndrome protein and huntingtin also influence synaptic neuropeptide stores by regulating direction-specific DCV capture (Cavolo et al, 2016;Bulgari et al, 2017).…”

Transmission, Development, and Plasticity of Synapses

“…Many of these mechanistic differences in DCV biology have been probed at Drosophila synapses using transgenic animals expressing an artificial neuropeptide-GFP-tagged rat atrial natriuretic factor peptide (ANF-GFP) (Rao et al 2001a). This transgenic line has proved highly valuable for DCV biology, revealing mechanisms of activity-dependent recruitment of DCVs (Shakiryanova et al 2005), DCV capture at active terminals (Wong et al 2012;Bulgari et al 2014), presynaptic ER-dependent Ca 2+ release driving DCV fusion (Shakiryanova et al 2007(Shakiryanova et al , 2011, and partial depletion of DCV contents upon release (Wong et al 2015). In addition to aspects of DCV release and trafficking, studies have revealed the role of the basic helix-loop-helix transcription factor Dimmed in driving neuroendocrine cell fate, including its role in regulating the expression of specific DCV proteins (Hewes et al 2006;Hamanaka et al 2010;Park et al 2011Park et al , 2014.…”