Activity Induces Fmr1-Sensitive Synaptic Capture of Anterograde Circulating Neuropeptide Vesicles

Abstract

Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Here experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins.

Significance statement: Synaptic release of neuropeptides and neurotrophins depends on presynaptic accumulation of dense-core vesicles (DCVs). At rest, DCVs are captured bidirectionally as they circulate through Drosophila motoneuron terminals by anterograde and retrograde transport. Here we show that activity stimulates further synaptic capture that is distinguished from basal capture by its selectivity for anterograde DCVs and its inhibition by overexpression of the fragile X retardation protein Fmr1. Fmr1 dramatically lowers DCV numbers in synaptic boutons. Therefore, activity-dependent anterograde capture is a major determinant of presynaptic peptide stores.

Keywords: SPAIM; axonal transport; peptidergic transmission; secretory granule; synaptic capture.

Figure 1.

Anterograde DCVs contribute to activity-dependent capture. A, Design of SPAIM experiment to detect contribution of anterograde DCVs. The axon was continuously photobleached (PB) while the most proximal bouton in the ROI was imaged. B, Pseudocolor images from a single experiment showing neuropeptide content before, immediately after 1 min of 70 Hz stimulation (Stim), and 4 min later. Scale bar, 2 μm. C, Quantification of the effect of upstream PB on stimulation responses. Bar indicates 70 Hz stimulation. For both controls (Con) and PB experiments, n = 5.

Figure 2.

Activity-dependent capture does not require retrograde DCVs. A, Design of SPAIM experiments to detect contribution of retrograde DCVs. B, Release and rebound due to capture are evident with continuous downstream photobleaching. n = 7. C, Release and capture persist after inhibiting retrograde transport with MB. DMSO control, n = 5; MB, n = 6. Bars, 70 Hz stimulation.

Figure 3.

Activity selectively increases capture of anterograde DCVs. A, DCV flux was measured in and out of the most proximal bouton in a branch following its photobleaching. Anterograde capture efficiency was calculated as (A_in − A_out)/A_in, while retrograde capture equaled (R_in − R_out)/R_in. B, Effect of stimulation (Stim) on anterograde capture efficiency (n = 13, p < 0.0001, paired t test). C, Effect of stimulation on retrograde capture efficiency (n = 13). Paired t test showed that there was no significant change.

Figure 4.

Fmr1 effects on type-Ib boutons and DCVs. A, Images showing Control (Con) and Fmr1 overexpressing type-Ib boutons with DCVs labeled with Dilp2-GFP (left), SSVs labeled with FM4-64 (center), and mitochondria (Mito) labeled with MitoTracker Red (right). Dashed lines show outline of boutons. Scale bars, 2 μm. B, Fmr1 increases bouton area. Con, n = 59; Fmr1, n = 73. C, Fmr1 decreases total Dilp2-GFP fluorescence per bouton (F_bouton) measured in confocal image stacks. Con, n = 59; Fmr1, n = 73. D, Fmr1 decreases individual DCV fluorescence (F_DCV). Con, n = 138; Fmr1, n = 114. E, Effect of Fmr1 on DCV flux through proximal boutons. Con, n = 8; Fmr1, n = 9. *p < 0.05; ***p < 0.001, ****p < 0.0001, unpaired t test.

Figure 5.

Fmr1 overexpression does not alter constitutive capture efficiency in type-Is boutons. A, Left, Representative images of type-Is boutons in control (Con) and Fmr1-overexpressing NMJs. Right, Quantification of bouton fluorescence. Con, n = 44; Fmr1, n = 40. ****p < 0.0001, unpaired t test. B, Capture efficiency measurements were made as in Figure 3. Con, n = 9; Fmr1, n = 12.

Figure 6.

Fmr1 overexpression inhibits activity-dependent capture. A, Data from type-Is boutons. Control (Con), n = 6; Fmr1, n = 8. B, Data from type-Ib boutons. Con, n = 4; Fmr1, n = 10. Bars, 70 Hz stimulation.

Figure 7.

Futsch/Map1B does not affect activity-dependent DCV capture or flux. A, Macroscopic release and capture responses to stimulation of type-Ib boutons in the Futsch N94 mutant. Bar, 70 Hz stimulation. B, DCV flux is unaffected in Futsch N94 boutons. Control (Con), n = 8; Futsch N94, n = 6.