Synaptic neuropeptide release by dynamin-dependent partial release from circulating vesicles

Abstract

Neurons release neuropeptides, enzymes, and neurotrophins by exocytosis of dense-core vesicles (DCVs). Peptide release from individual DCVs has been imaged in vitro with endocrine cells and at the neuron soma, growth cones, neurites, axons, and dendrites but not at nerve terminals, where peptidergic neurotransmission occurs. Single presynaptic DCVs have, however, been tracked in native terminals with simultaneous photobleaching and imaging (SPAIM) to show that DCVs undergo anterograde and retrograde capture as they circulate through en passant boutons. Here dynamin (encoded by the shibire gene) is shown to enhance activity-evoked peptide release at the Drosophila neuromuscular junction. SPAIM demonstrates that activity depletes only a portion of a single presynaptic DCV's content. Activity initiates exocytosis within seconds, but subsequent release occurs slowly. Synaptic neuropeptide release is further sustained by DCVs undergoing multiple rounds of exocytosis. Synaptic neuropeptide release is surprisingly similar regardless of anterograde or retrograde DCV transport into boutons, bouton location, and time of arrival in the terminal. Thus vesicle circulation and bidirectional capture supply synapses with functionally competent DCVs. These results show that activity-evoked synaptic neuropeptide release is independent of a DCV's past traffic and occurs by slow, dynamin-dependent partial emptying of DCVs, suggestive of kiss-and-run exocytosis.

FIGURE 1:

Dynamin enhances activity-dependent synaptic neuropeptide release. (A) Pseudocolor images of Dilp2-GFP in WT motoneuron synaptic boutons before and after 60 s of activity at 22°C (top) or 30°C (bottom). Bar, 2 μm. (B) Quantification of activity-induced release by WT boutons at 22°C (N = 7) and 30°C (N = 5). Note absence of temperature effect. (C) Pseudocolor images of Dilp2-GFP in shi^ts1 motoneuron synaptic boutons before and after 60 s of activity at 22°C (top) or 30°C (bottom). Bar, 2 μm. (D) Quantification of the effect of temperature on synaptic peptide release at shi^ts1 NMJs (N = 10 for 22^oC; N = 11 for 30^oC). *p < 0.05.

FIGURE 2:

Activity evokes partial emptying of DCVs in the nerve terminal. (A) Pseudocolor images of single Dilp2-GFP DCVs in type Ib boutons, which are outlined in white in left images, before (Pre) and after 60 s. (i) DCV in an unstimulated terminal; (ii) unresponsive DCV localized in a bouton that was stimulated for 60 s; (iii) responsive DCV in a bouton that was stimulated for 60 s. Note the drop in signal for iii only. Bars, 1.75 μm (bouton images on the left), 0.5 μm (magnified DCV images). (B) Histogram quantifying the range of responses to stimulation in 33 DCVs. No DCV released more than half of its neuropeptide content.

FIGURE 3:

Kinetics of neuropeptide release from single DCVs. (A) Time courses from single Dilp2-GFP DCVs. Horizontal bar, 20 s; vertical bar, 25%. Dotted line indicates start of 70-Hz stimulation. Release is plotted as a positive (upward) change. (i) Data from an unstimulated synapse; (ii–v) Data from stimulated synapses. (B) Ensemble-average time course from 33 DCVs. Curve is a single-exponential fit of data (τ = 21.5 s). (C) Average response from seven boutons. Curve is a single-exponential fit of data (τ = 17. 6 s). Horizontal bars in B and C indicate 45 s of 70-Hz stimulation.

FIGURE 4:

A single DCV can release twice. (A) Ensemble average from Dilp2-GFP DCVs stimulated twice for 10 s at 70 Hz (indicated by thick horizontal bars). Data are from six DCVs that displayed stepwise responses to the first tetanus. (B) Single-DCV time course with two stepwise responses to the double stimulation protocol in A. (C) Time courses of single Dilp2-GFP DCVs responding twice during a single prolonged tetanus (indicated by thick horizontal bar). Both responses were relatively large in i and small in ii. Horizontal dashed lines distinguish the baseline and signal after the first release event. Thin horizontal bars, 10 s; thin vertical bars, 10% release.

FIGURE 5:

Comparable release from visiting and captured DCVs. At the onset of tetanic stimulation, visiting DCVs had been present in the bouton for <1 min, whereas captured DCVs had been present >2 min. (A) Release probability for 26 captured and 16 visiting ANF-GFP DCVs. (B) Time courses of release from 19 captured (filled circles) and 8 visiting (open circles) ANF-GFP DCVs.

FIGURE 6:

Single-DCV release is similar in proximal and the most distal boutons. (A) Single ANF-GFP DCV release probability in the most distal bouton (Dist, N = 7) and proximal boutons 2–4 (Prox, N = 10). (B) Ensemble-average time course from five distally (closed circles) and eight proximally (open circles) captured ANF-GFP DCVs. Data were time binned in 5-s intervals. All DCVs here arrived by anterograde transport.

FIGURE 7:

DCVs delivered by anterograde and retrograde transport are functionally equivalent. (A) Single ANF-GFP release probability for DCVs that entered boutons by anterograde (Antero, N = 23) or retrograde (retrograde, N = 16) transport. (B) Ensemble-average time course from 16 anterograde (filled circles) and 11 retrograde (open circles) ANF-GFP DCVs. Data were time binned in 5-s intervals.