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First published online 9 December 2008
doi: 10.1242/jcs.034603

Journal of Cell Science 122, 75-82 (2009)
Published by The Company of Biologists 2009
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Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events

Xiaofeng Xia1, Volkmar Lessmann2 and Thomas F. J. Martin1,*

1 Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA
2 Institute of Physiology, Otto-von-Guericke-University, Magdeburg, 39120 Magdeburg, Germany


Figure 1
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  		Fig. 1. ANF-EGFP marks a subgroup of DCVs in cultured hippocampal neurons. (A) Single ANF-EGFP-labeled DCVs visualized by TIRF microscopy. Left panel: vesicular localization of ANF-EGFP in both the soma and neurites of hippocampal neurons. Right panel: fluorescent beads 100 nm and 400 nm in diameter visualized under the same TIRF settings. (B) Immunocytochemical characterization of ANF-EGFP puncta. Row 1: all ANF-EGFP-containing vesicles were positive for the DCV marker SgII. Row 2: all ANF-EGFP-containing vesicles contained the Ca2+ sensor SytI. Row 3: ANF-EGFP-containing vesicles were distinct from monoaminergic VMAT2-positive vesicles. Row 4: most ANF-EGFP-containing vesicles also contained immunoreactive BDNF. Fluorescence line scans are shown in insets. These indicate that all ANF-EGFP-containing vesicles (green) also contained SgII and SytI (red). All images were visualized by TIRF microscopy. Scale bars: 1 µm.

 

Figure 2
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  		Fig. 2. Time course of the elevation of intracellular Ca2+ level by K+ stimulation revealed by Fluo-4 imaging. Cells were stimulated at 90 mM K+ (red) or 30 mM K+ (blue).

 

Figure 3
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  		Fig. 3. Exocytosis of ANF-EGFP-containing DCVs in neuronal soma and neurites. (A) Comparison of release probabilities for vesicles in soma and neurites. In the soma, about one third of the plasma-membrane-proximal vesicles underwent exocytosis in 100 seconds, whereas less than 1% in the neurites could release. A total of 1091 vesicles were analyzed in soma and 1344 vesicles in neurites (***P<0.001). (B) Depolarization-evoked DCV events exhibit long latencies. Events in the soma and neurites were binned in 10-second intervals and plotted by percentage. Soma events were fitted with a single exponential curve (broken line) to calculate a time constant, {tau} (15.6 seconds). Neurite events did not fit a single exponential curve but were evident at long times. (C) Single exocytosis events in neuronal soma. (Ci) A representative example of DCV exocytosis in the soma. The time interval between two consecutive points was 0.1 seconds. The time between the fluorescence baseline and the fluorescence peak (fluorescence rise time) was designated as tr. Release of content was calculated based on the fluorescence change ({Delta}F) between 2 seconds before and 15 seconds after exocytosis. (Cii) A DCV in the soma underwent two consecutive exocytic events. Shown below the curves are images of the corresponding DCV at a time interval of 0.2 seconds. Start points of the events are indicated by arrows. Scale bars: 500 nm. (D) A representative example of DCV exocytosis in the neurites. The time interval between two consecutive points was 0.2 seconds. Shown below the curve are images of the corresponding DCV at a time interval of 0.2 seconds. The start point of the exocytic event is indicated by an arrow. Scale bar: 500 nm. (E) Analysis of the content released during exocytosis for DCVs in soma and neurites. Content release was determined as the loss of fluorescence ({Delta}F) by subtraction of the value 2 seconds before an event from that obtained 15 seconds after the event. Gaussian fitting revealed two populations of events in the soma (solid lines) and one population in the neurites (broken line). (F) Comparison of the fluorescence rise time (tr) of the events in soma and neurites (***P<0.001). A total of 332 fusion events in soma and 48 events in neurites were analyzed in B, E and F.

 

Figure 4
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  		Fig. 4. Exocytosis of BDNF-EGFP-containing DCVs in neuronal soma and neurites. Exocytosis of BDNF-EGFP in the soma (A) and neurites (B) was imaged using methods similar to those used in Fig. 3. Exocytosis of BDNF-EGFP-containing DCVs was not accompanied by a fluorescence cloud or by a significant loss of fluorescence. Curves shown are averages of ten individual events with standard deviations indicated. Shown below the curves are images of representative DCVs at 1.0-second time intervals. Start points of the exocytic events, occurring at various times following depolarization, are indicated by arrows.

 

Figure 5
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  		Fig. 5. DCV exocytosis in neuronal soma under conditions of strong and weak depolarization. (A) Comparison of release probabilities at high [total number of analyzed vesicles (n)=1091] and low (n=1525; ***P<0.001) K+ stimulation. (B) A representative example of DCV exocytosis in soma under low K+ (30 mM) stimulation. The time interval between two consecutive points is 0.1 seconds. (C) Comparison of the fluorescence rise time (tr) of events at high- and low-K+ stimulation (***P<0.001). (D) Analysis of the content released during exocytosis for DCVs in soma at high (90 mM) and low (30 mM) K+ stimulation. The fluorescence loss ({Delta}F) from a vesicle was calculated from values 2 seconds before and 15 seconds after an event. Gaussian fitting revealed two populations under both stimulation conditions. At low-K+ stimulation (broken lines), a larger subset of DCVs exhibited extensive content release compared with DCVs stimulated at high K+ (solid lines). A total of 332 fusion events under 90 mM K+ stimulation and 90 events under 30 mM K+ stimulation were analyzed in C and D.

 

Figure 6
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  		Fig. 6. L-type Ca2+ channels mediate DCV exocytosis in hippocampal neurons. Release probabilities in the neuronal soma in response to 90 mM K+ were determined after treatment with the indicated Ca2+-channel inhibitors. Verapamil at 100 µM (specific for L-type Ca2+ channels) completely blocked evoked DCV exocytosis [total number of analyzed vesicles (n)=803], whereas 10 µM {omega}-conotoxin MVIIC (N- and Q-type specific, n=734) and 200 nM {omega}-agatoxin TK (P-type specific, n=712) were without effect (P>0.5 by t-test).

 

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