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.

Keywords: LDCV; nerve terminal; neurotransmission; secretory granule.

Fig. 1.

Type Ib and type III boutons differ in neuropeptide content and DCV number. (A) Images of type Ib (Upper) and type III (Lower) boutons expressing Anf-GFP. (Scale bars, 2 μm.) (B) Neuropeptide fluorescence per bouton (F_bouton) of type Ib (n = 16) and type III boutons (n = 20) in arbitrary units (au). **P < 0.01. (C) Depolarization-evoked neuropeptide release in type Ib (n = 8) and type III boutons (n = 4) measured as the percent decrease in fluorescence. ns, not significant. Boutons were stimulated with elevated extracellular K⁺ for 3 min. (D) Single DCV fluorescence in axons leading to type Ib (n = 56) and type III boutons (n = 53). ****P < 0.0001. Error bars represent SEM.

Fig. 2.

DCV transport and turnover are similar in type Ib and type III boutons. (A) Anterograde (open bars) and retrograde (filled bars) DCV flux at proximal type Ib (n = 19) and type III boutons (n = 23). ns, not significant. (B) The percent FRAP over time following photobleaching in type III boutons (n = 4). (C) Age-dependent labeling of DCVs expressing Anf tagged with mK-GO in type Ib and III boutons. For each bouton type, green (young) and red (old) images are shown side by side. (Scale bars, 2 μm.) (D) Ratio of green to red fluorescence in type Ib (n = 8) and type III boutons (n = 10).

Fig. 3.

DCV capture in type III boutons is efficient. (A) Time-lapse images of type Ib and type III boutons before and after photobleaching boutons. FRAP shows DCV accumulation is marked in the most distal type Ib boutons and the most proximal type III boutons. Contrast was adjusted to visualize puncta. Numbers indicate time in minutes. Proximal unbleached boutons are shown at the top of images. (Scale bar, 2 μm.) (B) DCV anterograde flux into and out of photobleached proximal type III boutons shows high capture efficiency in type III boutons (n = 7). **P < 0.01. (C) Change in neuropeptide content after a 3-min depolarization (indicated by arrow) of type III boutons (black circles) (n = 8). Data are normalized to the initial decrease in fluorescence (ΔF). Dotted curve (open circles) shows the rebound in type Ib boutons (replotted from ref. 17) evoked by the same stimulus that is indicative of activity-dependent DCV capture.

Fig. 4.

DIMM confers peptidergic type III bouton properties onto type Ib boutons. (A) Fluorescent images of type Ib boutons on muscle 1 in wild-type (Left) and DIMM-expressing (Right) flies. (Upper) Anf-GFP in green. (Lower) The same boutons stained with TRITC-HRP antibody (red) to reveal presynaptic morphology. (B) FRAP measured as percent recovery in 15 min for muscle 1 type Ib boutons with and without DIMM. Note the switch from distal to proximal recovery. (Scale bars, 2 μm.) (C) Quantification of FRAP in muscle 1 type Ib boutons (n = 5), type III boutons (n = 8), and type Ib boutons expressing DIMM (n = 6). *P < 0.05, **P < 0.01.