Full text

Turn on search term navigation

ARTICLE

Received 27 Apr 2011 | Accepted 5 Oct 2011 | Published 8 Nov 2011 DOI: 10.1038/ncomms1534

Extrasynaptic vesicle recycling in mature hippocampal neurons

Arjuna Ratnayaka1,*, Vincenzo Marra1,*, Tiago Branco2,3 & Kevin Staras1

Fast neuronal signalling relies on highly regulated vesicle fusion and recycling at specialized presynaptic terminals. Recently, examples of non-classical neurotransmission have also been reported, where fusion of vesicles can occur at sites remote from conventional synapses. This has potentially broad biological implications, but the underlying mechanisms are not well established. Here we show that a complete vesicle recycling pathway can occur at discrete axonal sites in mature hippocampal neurons and that extrasynaptic fusion is a robust feature of native tissue. We demonstrate that laterally mobile vesicle clusters trafcking between synaptic terminals become transiently stabilized by evoked action potentials and undergo complete but delayed Ca 2+ -dependent fusion along axons. This fusion is associated with dynamic actin accumulation and, subsequently, vesicles can be locally recycled, re-acidied and re-used. Immunouorescence and ultrastructural work demonstrates that extrasynaptic fusion sites can have apposed postsynaptic specializations, suggesting that mobile vesicle recycling may underlie highly dynamic neuronneuron communication.

1 School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK.2 Wolfson Institute for Biomedical Research, University College London , London WC1E 6BT, UK.3 Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, UK. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to K.S. (email: [email protected]).

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

Most information transfer in the mature CNS relies on fast chemical transmission at synapses, ultrastructurally distinct neuron neuron apposition points at which a

presynaptic terminal harbouring a cluster of synaptic vesicles is located adjacent to a postsynaptic target. Th e specialized nature of these structures, which includes the complex arrays of proteins that regulate and mechanically facilitate the exocytic fusion of vesicles 1,

has led to the classical view that transmission occurs exclusively at such sites along the axon. In recent years, however, a new perspective on the independence of synaptic operation has emerged with novel examples of neuronal signalling that deviate from a simple point-to-point transmission model 2,3. Th ese include spillover, where transmitter released at one presynaptic terminal diuses away from the synaptic cle resulting in synaptic crosstalk and / or the activation of extrasynaptic receptors 4 , and ectopic transmission 2 , where vesicle fusion and transmitter release occur at sites away from anatomically dened presynaptic active zones 513 . In hippocampal neurons, a further departure from the classical model of synaptic transmission has come with the nding that recycling synaptic vesicles can be highly mobile, both in mature cultured neurons 1418 and native tissue 17, moving readily between synaptic release sites and participating in vesicle fusion at new synaptic hosts. As such, individual terminals essentially form part of a larger vesicle superpool 17,18 with important potential implications for axonal synapse synapse interactions 17,19,20.To date,

attention has focused on the impact of mobile vesicles on recipient synaptic terminals 15,17, but evidence also indicates that traffi cking vesicles can be fusion-competent during transit, a property rst reported in developing neurons 2127 but since demonstrated in older cells 16,17. Given the large number of traffi cking vesicles moving along axons between established synapses 17 , axonal fusion in mature neurons could represent an important extra pathway for neuronal signalling.

Here we have used a combination of exocytic and endocytic reporters of vesicle recycling, Ca 2+ imaging and correlative uorescence and electron microscopy to characterize the dynamics and mechanisms underlying extrasynaptic vesicle fusion, both in native tissue and mature hippocampal neurons. We demonstrate that axonal segments of neurons in acute hippocampal slices can support fast fusion of mobile vesicles in response to action potentials. High-rate time-lapse imaging reveals the dynamics of this process: typically vesicles transiently stabilize before fusion and exocytose with fast release kinetics. Mobile vesicles can be recycled and reused outside synaptic terminals, and, using ultrastructural and uorescence data, we show that these sites can be anatomically specialized and lie adjacent to postsynaptic targets. Our ndings

oer new insights into the presynaptic mechanics underlying an extra feature of information transmission in central neurons that deviates from the conventional model of point-to-point transmission and could signal dynamic aspects of neuronal function.

ResultsMobile vesicle fusion at axonal sites. Recycling vesicles are highly mobile between synaptic terminals 1418 as evidenced in time-lapse sequences ( Supplementary Fig. S1 ). Importantly, these traffi cking vesicles can undergo fusion, both aer integration into a synaptic host 15,17 or orphan synapse 16 , but also at times immediately following 16 or even during transit 17 . To test the relevance of this in native tissue, we characterized stimulus-evoked vesicle dynamics in acute hippocampal slices. Synaptic terminals in CA1 were loaded with FM1-43 (refs 28, 29) by electrical stimulation (10 Hz 1,200 action potentials, AP) of Schaer-collaterals 30,31 with eld

potential recordings conrming eective stimulation of the target CA1 region ( Fig. 1a ). A er washing, uorescence imaging revealed robust punctate staining ( Fig. 1b ) that could be rapidly destained with electrical stimulation ( n=18 slices from 5 animals), where the rate of uorescence loss increased with stimulation frequency ( Fig. 1b,c ). Next, we looked for uorescent traffi cking puncta in time-lapse sequences 17 to test their stimulus-driven fusion-competence. Mobile packets were readily observed, both integrating into stable terminals ( Supplementary Fig. S2 ), but also traffi cking linearly along unlabelled lengths of intersynaptic axon ( Fig. 1d ). We found that stimulation appeared to stabilize these packets and evoked reliable dye loss ( Fig. 1e,f ) with the rate and extent of destaining in mobile vesicles comparable to stable synaptic neighbours (

for

stable: 145s,

for mobile: 15 7s, n=15 and 11 from 3 slices from 2 animals, P=0.93, t-test, Fig. 1f).

Next, we turned to FM1-43-labelling in dissociated cultures, where we could characterize the processes that support extrasynaptic fusion in detail. We restricted our analysis to discrete packets that were mobile at the time of stimulation, remained at intersynaptic sites during the whole stimulation period and showed clear activity-evoked dye loss ( Fig. 2a ). To characterize the relationship between activity and vesicle movement, we generated kymographs for target packets ( Fig. 2b,c ) and measured the average rate of vesicle movement immediately preceding and following stimulus onset (40 Hz 1,200 AP). Again, we found a signicant average reduction in mobility following the stimulus ( Fig. 2d ). Thus, overall, acti vity tends to result in stabilization of previously mobile packets at sites between conventional terminals. Nonetheless, it was clear

d e f

CA1S R 18 s 0 s

20 s 300 s

Stable

Stim

100

34 s

100

Mobile

2 Hz10 Hz20 Hz

120

10 Hz

120

10 Hz

CA3 DG

0 100 200

0 60

Figure 1 | Mobile vesicle fusion in acute slice. ( a) Preparation used and electrical response to stimulation. FM-dye-loading of terminals in the stratum radiatum of CA1 is achieved by electrical stimulation (S) of Schaffer collaterals. Typical eld excitatory postsynaptic potential recordings (grey lines) from target CA1 region (R) in the presence of AP5 are shown, with average trace in red. Vertical line on left shows the end of the stimulus artefact (stimulus:1 ms). Red arrow is bre volley. Black arrow is fEPSP. Vertical bar: 0.5 mV, Horizontal: 2 ms. ( b) Sample images of uorescent puncta and destaining at 10 Hz after 0 s. Scale bar, 2 m. (c) Single exponential curve ts from average data showing stimulation-evoked dye loss at 2, 10 and 20 Hz (16, 47 and 19 synapses, respectively from 1 slice each from 2 animals). ( d) Sample time-lapse images and kymograph showing FM-dye packet mobility and destaining. Red triangles indicate 10 Hz stimulation. Scale bar, 5 m. Vertical bar, 10 s. ( e) Destaining curve traces for packet from ( d) (red) and seven stable neighbouring synapses (blue). ( f) Summary destaining curves for mobile packets ( n=11) and stable synaptic neighbours (n=15).

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

ARTICLE

16 0

8 10 48 s

SytICypHer5E

Pre +40 s

103

222

20 Hz

30 20 10 10

40 Hz

0.8

pix per s

120 40 Hz

120

40 Hz

100

0 Bef Aft

30 Hz

100

20 Hz

120 40 Hz

120 20 Hz

r m

100

%Fluorescence

0 4 8

Stable

[afii9848](s)

Mobile

Stab

Mob

0 10 20 30 40 50

0 30

s s

Figure 2 | Stimulation-evoked movement and fusion properties of mobile vesicles. ( a) Time-lapse of axon of FM1-43-loaded neuron with uorescently labelled stable terminals (orange arrowheads) and mobile packet (white arrowhead). Stimulation results in rapid dye loss. Scale bar, 1 m.

( b) Kymograph generated from a line scan along the axon of the same sequence from ( a). Green line indicates prole of mobile packet. Scale bar, 1m. Horizontal: 20s. (c) Second example kymograph with a stable synapse and two mobile packets (green lines). Scale bar, 1 m. Horizontal: 20 s.

( d) Histogram showing mean s.e.m. mobility rates for trafcking packets before and after stimulation (pixels per sec before: 0.62 0.11, after: 0.120.04, paired t-test,

P<0.01, n=12 from 6/2). (e) Histogram showing % occurrence of three different categories of stimulation-evoked movement dynamics: s, stabilized; r, reduced trafcking rate; m, movement at pre-stimulation rate, for n=46 from 12/7. (f,g) Mean s.e.m. FM1-43 destaining plots ( g)

for mobile vesicle packets ( n=13 from 6/2) and stable terminals from adjacent regions (n=60). Lines are single exponential ts. inset: Mean s.e.m. destaining plots using faster acquisition imaging reveals delayed dye loss in mobile packets versus stable synapses ( n=6 from 2/1). (f) Means.e.m. for single exponential curve ts from ( g). (h) Sample destaining plots for two mobile packets (red lines), one moving at stimulation point (top) and one that spontaneously stabilized before stimulation (bottom). Neighbouring stable synapse traces are shown in blue. ( i) SytI-CypHer5E (loading: 600 APs 10 Hz, left, Scale bar, 5 m.) and stimulation-evoked quenching (right, Scale bar, 1 m). (j) Time-lapse sequence of mobile SytI-CypHer5E packets along axons (white arrowheads) quenching with stimulation. Scale bar, 1 m. (k) Mean s.e.m. plots of stimulation-evoked uorescence loss at stable terminals ( n=24 from 3/2) and mobile packets (n=6 from 3/2).

that at the level of individual packets, more complex relationships between mobility and stimulation could sometimes be observed. We investigated this by categorizing movements for n = 46 packets (12 coverslips from 7 culture preparations, or 12 / 7) across the range of experimental conditions used in this paper ( Fig. 2e ). Aer stimulation the majority of packets (61 % ) stabilized fully, but 28 % continued to mobilize at a reduced rate and 11 % moved at the same rate as before. To gain some insight into the incidence of mobile destaining, we next quantied the number of FM-dye-loss events occurring through mobile fusion in clearly dened axonal segments adjacent to stable synaptic boutons ( n = 50 from 4/2, length: 3.5m). Our analysis revealed that along these segments, 29.5 % of individual dye-loss events arose from mobile fusion. As such, this represents a substantial component of measured dye-loss events suggesting that this process has considerable potential relevance for signalling.

Next, we examined mobile vesicle fusion kinetics. 40 Hz stimulation produced reliable destaining of mobile packets and stable terminals in the same eld of view ( Fig. 2f,g ). Th is was also conserved in very mature neurons (DIV32, Supplementary Fig. S2 ). Overall, dye loss for mobile and stable puncta had a similar prole but the destaining time course was signicantly slower for mobile packets (

for stable: 7 1s,

for mobile: 11

1s, P<0.01,

t-test,

n=13for

each from 6 / 2, Fig. 2f ). We attributed this to delayed onset of dye loss, revealed in pairwise comparisons for the rst 6 seconds aer stimulation (stable versus mobile, 2 s: 0.80

0.03 and 0.910.03, P<0.01; 4s: 0.630.04 and 0.760.03, P<0.01; 6s: 0.540.04 and0.680.04, P<0.01, t -tests) and supported by further experiments using faster acquisition imaging to visualize the initial destaining period ( Fig. 2g , stable versus mobile, 1 s: 0.90 0.01 and 1.020.01, P<0.01; 2s: 0.810.01 and 0.920.01, P<0.03; 3s: 0.750.01 and0.890.02,

P<0.01, t-tests,

n=6foreach from 2/1).We reasoned

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

this lag might correspond to a period required for vesicles to move to and / or become associated with an appropriate section of target membrane. To test this, we examined destaining proles of mobile packets that had spontaneously stabilized several frames before stimulation. Th ese packets underwent activity-evoked dye loss without delay when compared with destaining proles from neigh-bouring control synapses (pairwise comparisons for rst 3 points aer stimulation, stable versus stabilized packet, 2 s: 0.84 0.02 and 0.860.01; 4s: 0.710.06 and 0.730.03; 6s: 0.610.06 and0.580.04, P>0.25, paired

t -test for all, n=4 for each from 3/2 (Fig. 2h). Th us, steps required to change mobile vesicles into fusion-competent vesicles could be suffi cient to account for a delayed onset to exocytosis.

To conrm our ndings, we next tested an alternative vesicle reporter, SytI-CypHer5E, based on a uorescent-conjugated antibody targeted to the luminal domain of the vesicle protein synaptotagmin 32. CypHer5E is pH-sensitive, uorescing strongly in the acidic vesicle lumen, but quenched when the uorophore is exposed to neutral extracellular pH ( Fig. 2i ). Th us it provides a sensitive readout of intact, recently recycled, acidied vesicles. As expected, SytI-CypHer5E puncta underwent stimulation-evoked uorescence quenching as vesicles fused with the membrane ( Fig. 2i ). Moreover, time-lapse imaging sequences revealed laterally mobile packets of vesicles that underwent stimulation-evoked uorescence loss (

for stable: 72s,

for mobile: 12

2s,

n=24 and

n=6 from 3/2, P=0.08, t-test, Fig. 2j,k). Th ese results, using a dierent vesicle probe, support our FM-dye ndings.

Th e experiments described so far have relied on recycling vesicle probes that label functional vesicle pools. Next, we considered whether axonal sites supporting mobile fusion might, in fact, be presynaptically silent synapses containing non-recycling vesicles that are functionally engaged by the arrival of recycling ones. We performed dual-imaging time-lapse experiments using FM1-43 and synaptophysinI-mCherry (SypI-mCherry) ( Fig. 3a ), a non-selective marker of vesicles, to examine the specic dependency of a site where mobile fusion takes place on the prior presence of mCherry uorescence. Although, as expected, SypI-mCherry and FM-uorescence were co-localized in traffi cking packets, mobile vesicle fusion events could readily occur at sites that were previously SypI-mCherry-negative ( Fig. 3a,b ). Th us, target fusion sites without pre-existing pools of vesicles can support mobile exocytosis.

Axonal fusion sites can support endocytosis. Next, we tested whether extrasynaptic fusion sites can support endocytosis and local vesicle reuse. SytI-CypHer5E is an informative tool to address this question, because it reports both the exocytic phase of vesicle turnover, but also the endocytic leg as a recovery in uorescence signal when retrieved vesicles are re-acidied and CypHer5E uorescence becomes unquenched ( Fig. 4a,b ). Although the high sensitivity of CypHer5E to photobleaching precluded a detailed analysis of endocytic properties of mobile vesicles, uorescence recovery aer stimulation was seen ( Fig. 4b,c ). Th us, a mobile vesicle packet can undergo fusion, endocytosis and re-acidication at an axonal site distinct from a conventional terminal, and then become mobilized again. Next, we examined this with SypHyx2, that reports vesicle exocytosis with a rise in uorescence and quenches when recycling vesicles become re-acidied aer endocytosis 33 (Fig. 4d,e). We found that stimulation could bring about an initial increase in uorescence signal in these packets ( Fig. 4e ) reecting vesicle fusion and consistent with our FM-dye data. Moreover, conrming our SytI-CypHer5E ndings, we also recorded a subsequent signal reduction, corresponding to endocytosis and re-acidication ( Fig. 4d,e ). Overall, the average time course of endocytosis from all mobile packets was comparable to stable control synapses (

for

stable:46s,

for mobile: 49 s, n=40and n=7from 3/3,Fig.4d) suggesting that the two groups shared similar underlying endocytic

mechanisms. Taken together, our ndings show that mobile vesicles can undergo a complete recycling pathway typically correlated with a stabilization of mobile vesicles ( Fig. 4e ). Finally, we examined whether packets could undergo multiple rounds of fusion. Neurons were subjected to a paired stimulation protocol evoking a reliable double rise in uorescence in stable synapses and mobile packets ( n=3from 3/2,Fig.4f,g),supporting theideathat mobilevesicles could engage in repeated vesicle recycling events.

Dynamic recruitment of actin to sites of axonal fusion. Actin is an important and highly dynamic cytoskeletal element that is concentrated at synapses 34,35 ( Fig. 5a ), and implicated as a synaptic scaolding element supporting vesicle turnover 35 , in synaptic remodelling 34 and in lateral vesicle traffi cking 15,18. We reasoned that actin remodelling might accompany or even facilitate the vesicle movement along axons, and support recycling events. To test this, we labelled GFP-actin-expressing neurons with the vesicle marker SytI-Oyster550 ( Fig. 5a ) and monitored dynamic movements with dual-colour time-lapse imaging. Vesicle movements to new sites along the axon were associated with directed recruitment of actin ( Fig. 5b ) but aggregation followed, rather than preceded, the mobilization of vesicle packets ( Fig. 5b,c ). Moreover, fusion-competence of mobile vesicles did not appear to be critically dependent on the presence of actin because eective activity-evoked dye loss could occur before signicant actin condensation (Fig. 5d,e).

Postsynaptic targets and ultrastructure. Mobile fusion will have particular relevance, if recycling can occur at sites apposed to post-synaptic targets. To examine this, we combined FM-dye destaining with co-labelling for postsynaptic markers. In our cultures, stable puncta appeared to be co-apposed to postsynaptic targets in nearly all cases (glutamate receptor 1 (GluR1): 90 3%, n=920 synapses,4/1;PSD-95:913%,

n=999synapses,3/1;GABA(-aminobutyric acid)-A-R: 357%, n=752 synapses, 5/2). Mobile fusion events onto target structures were readily observed (GluR1 or GluR2, n=20examplesfrom 6/2;GABA-A-R, n=5from 3/2)(see

sypI FM1-43

Stable synapse

Mobile packet

2 4 8 12

40 Hz

Norm. F

Pre

Post

Pre

Post

Pre

Post

Pre

Post

0 FM1-43 SypI SypI

FM1-43

Figure 3 | Fusion occurs at previously vesicle-free sites along axons. ( a) Example time-lapse imaging experiment of FM1-43 uorescence in SypI-mCherry-expressing neuron, showing that mobile fusion (white arrowhead) can take place at novel sites not already populated with vesicles. Yellow ovals indicate axonal site of destaining. Scale bar, 1 m.

( b) Quantication of experiments in ( a). Histograms show FM1-43 (green) and SypI-mCherry (red) uorescence at fusion sites for mobile packets and neighbouring stable terminals ( n=4 from 3/3). Pre corresponds to the uorescence for each region at the time point where mCherry signalis at its lowest for the mobile packet. Post represents the time point after the packet has stabilized at the destaining site. All values are normalizedto post-uorescence values for each condition. The histogram shows that before the arrival of the mobile packet at the axonal site, FM1-43 and mCherry uorescence are close to background levels. Values are means.e.m.

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

ARTICLE

SytICypHer5E

20 Hz

10 0 20 40 60 100 120 s

17 19 21 23 41 48 75 s

120

Pre

180 s

20 Hz

Mobile Stable

0 80

20 140 s

20 Hz

20 Hz 20 Hz

20 Hz

9 6 3 14 65 129 s

120

20 Hz

Mobile Stable

120

100

Stable

Mobile

0 20 40 s

0 150

50 100

20 Hz

Figure 4 | Axonal fusion sites support vesicle endocytosis, re-acidication and reuse. ( a,b) Image sequence and plot of CypHer5E uorescence showing recovery of signal after stimulation (400 APs, 20 Hz) at a stable synaptic terminal ( a) and means.e.m. uorescence plot ( b) reecting the endocytosis and re-acidication of vesicles ( n=13 stable synapses from 1 coverslip). Scale bar, 1m. Note the change in timescale along x axis after stimulation. Red points are associated with recovery of the mobile packet from (c) ( n=1 from 1 coverslip). (c) Time-lapse of a SytI-CypHer5E-labelled mobile vesicle packet (white arrowheads) showing mobility, stabilization, activity-evoked fusion, re-acidication and remobilization. Scale bar, 1 m. (d,e) Endocytosis in mobile packets examined using SypHyx2. ( d) Summary plot of responses in mobile packets ( n=7 from 3/3, red) and stable terminals (n=40, blue). Points are means.e.m. (e) Sample time-lapse sequence showing mobile packet (white arrowhead) and neighbouring stable terminal (orange arrowhead) exhibiting a uorescence rise on stimulation (0 15 s). Scale bar, 1 m. (f) Sample time-lapse frames showing a mobile packet (white arrowhead) as well as a stable synapse (orange arrowhead) undergoing multiple fusion events with two rounds of 20 Hz stimulation. Scale bar, 1 m. (g) Mean plots showing responses to dual stimulation protocol for mobile packets ( n=3, from 3/2, red) and their neighbouring stable terminals (n=3, blue). For clarity, s.e.m. error bars are not shown.

Fig. 6a-d ). Notably, a variety of dynamic relationships were apparent between mobile packets and their targets. For example, postsynaptic markers could be presynaptically unapposed before the arrival of a packet ( Fig. 6b ) or, alternatively, host other transient presynaptic traffi c ( Fig. 6c ). Also, evoked dye loss could be delayed until a mobile packet traffi cked to a postsynaptic site ( Fig. 6c , middle). Although mobility of synaptic vesicles was high, coordinated lateral movement of postsynaptic label was not seen ( Fig. 6e,f ). Consistent with previous work 16 , we also observed some mobile packets that destained at sites that did not seem to be intimately associated with specic postsynaptic markers although these were more conned to isolated processes in sparse regions of the culture.

To gain further insight into the structural organization of mobile fusion sites, we next subjected fusion-competent traffi cking vesicles to ultrastructural analysis. During imaging, FM-dye-labelled synapses were partially destained via eld stimulation (120 AP, 30 Hz, Fig. 7a,b ), then immediately xed and photoconverted so that FM-dye-containing vesicles become electron-dense ( Fig. 7a ) 3638.

Imaging revealed fusion-competent mobile packets ( n=4 from 1/1,

Fig. 7c ) that we could target in the electron microscope using correlative methods outlined previously ( Fig. 7d-f ) 15,39 . At the ultrastructural level, all target packets consisted of a loosely arranged cluster of photoconverted and non-photoconverted vesicles, corresponding to recycling and non-recycling vesicles, respectively ( Fig. 7g i , Supplementary Fig. S3 ). Th e mean percentage of recycling vesicles relative to total vesicles was 34

9%(n=4).Notably,allana-

lysed packets contained docked vesicles (mean: 1.5 0.5, n=4) and were along an axonal section that was intimately apposed to a post-synaptic structure ( Fig. 7g ), showing that these were not orphan sites. Th us, mobile vesicle clusters can fuse at regions of the axon that have many structural features found in conventional terminals, and would therefore be relevant for neuron neuron signalling.

Action potential dependent Ca 2+ entry at axonal sites.Exocytosis requires a rapid increase in local [Ca 2+ ] (ref. 40). However, Ca 2+ increases along the axon are known to be smaller and slower than in synaptic terminals 41,42, raising questions about how axonal sites could support vesicle fusion. To address this, we rst conrmed the dependency of mobile vesicle fusion on the presence of extra-cellular Ca 2 + by quantifying the stimulation-evoked dye loss in mobile vesicle packets ( % dye loss corrected for photobleaching a er 600 APs, 2.5 mM,: 45 8%, n = 6 from 2/1; 0 mM: 4 3%, n = 10 from 2/1, t-test,

P < 0.01). Next, we performed calcium imaging experiments to provide information on the kinetics of Ca 2 + entry along axonal segments between conventional terminals. We expressed the fusion construct SypI-mCherry to label presynaptic terminals and lled transfected neurons with the calcium indicator OGB-1 (200 M) via whole-cell patch clamp ( Fig. 8a ). We then targeted mCherry-positive synapses and their anking axon and imaged OGB-1 uorescence with line scans during single-action potentials evoked via the patch-pipette. As expected 41 , we recorded reliable and rapid rises in Ca 2 + at

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

Sytl Actin Merge

14 8 36 s

100

Sytl

Sytl Actin

Actin

0 2 4 13 s

0 10

Sytl Actin

20 Hz

14 8 36 s

Normalized F

Sytl

0 Actin

20 20

0 40

20 Hz

Figure 5 | Recruitment of actin to mobile vesicle packets. ( a) Images showing co-localization of SytI-Oyster550 (red) and GFP-actin (green) in actintransfected neurons. Scale bar, 1 m. (b) Sample time-lapse images showing movement of vesicle packet (red arrowheads) followed by aggregation of actin to the same site (green arrowheads) and corresponding uorescence proles. Scale bar, 1 m. (c) Summary mean s.e.m. data plots for vesicle movement events from n = 5 packets (red) and actin (green) from 2 coverslips from culture preparations. Data show average uorescence in the region of interest into which the packet moved. Data is normalized to peak in actin uorescence illustrating the kinetics of uorescence turnover. ( d) (Left) sample time-lapse images showing movement of a SytI-Oyster550-labelled vesicle packet (red arrowhead) to new site before undergoing activity-evoked vesicle fusion with 20 Hz stimulation, and corresponding images for GFP-actin (green). Scale bar, 1 m. (e) Plots for SytI-Oyster and actin from ( d). Activity-evoked dye loss is shown with single exponential t.

synaptic terminals, and also established that these signals were not saturated ( Supplementary Fig. S4 ). At sites along the anking axon, smaller and slower Ca 2 + changes could be observed, although these were still signicantly above baseline ( Fig. 8b ). Importantly, though, the distinction between Ca 2 + dynamics

at synaptic and non-synaptic regions was not always apparent. In 57 % of axonal segments ( n = 7 from 7/2 with an average length of 4.0 0.2 m), we also observed hotspots of fast and substantial Ca 2 + increases that were not correlated with stable SypI-mCherry positive puncta ( Fig. 8c e ). At these sites, the peak Ca 2 + rise was the same as at synaptic sites (1.02 0.15,

P > 0.05,

F/F0 normalized to neighbouring synaptic region, t-test,

n = 4

n = 5,

from 4 / 2, Fig. 8d,f ), whereas intermediate axonal sections were signicantly dierent (0.17 0.07, P < 0.01, t-test). Moreover, synap ses and hotspots had a similar fast rise time, and this was dierent to responses at intermediate axonal sites ( Fig. 8d,f ; synapse 11.32 1.76 ms; hotspot 12.67 1.82 ms; axon 28.322.99 ms,ANOVA(analysisof variance)F(2,9) = 17.38,

P < 0.001, Tukey s test S versus H, S versus A, H versus A; P > 0.05,

P < 0.01,

n = 4). On repeated stimulation, hotspots exhibited consistent responses suggesting that they were reliable loci for activity-dependent Ca 2 + entry ( Fig. 8e ). We also performed experiments using the lower affi nity calcium indicator, Fluo-5F on SypI-mCherry-transfected neurons. We identied hotspots in three out of six axonal segments (from 6 / 1) and, again, found peak, rise times and decay times, equivalent to those for stable synapses ( Supplementary Fig. S5 ).

Taken together, our ndings suggest that activity-evoked Ca 2+

rises occur throughout the axon but specic sites along axonal

segments can support the fast action potential-evoked Ca 2 + rises

necessary for axonal vesicle fusion. To test this directly, we carried out experiments using the membrane permeable form of OGB-1 (OGB-1-AM) in which we monitored stimulation-evoked Ca 2 + dynamics along axons followed immediately by time-lapse-

imaging of FM-dye-destaining at the same sites. We found that fusion-competent mobile packets ( n = 5 from 3/2) destained at sites where a discrete and substantial localized rise in Ca 2 + also

occurred, equivalent to those we characterized above. For two of these, we also carried out post-x immunolabelling and revealed a PSD-95-positive signal, consistent with the idea that fusion-sites can be relevant to postsynaptic signalling ( Fig. 8g ). Importantly, examples of mobile packets that did not undergo dye loss ( n = 5 from 3 / 2, mean dye loss for 5 frames a er stimulation: 3 1 versus 26 6% for fusion-competent packets, P < 0.05, t-test) were present at sites with a much smaller rise in Ca 2 + signal than for fusion-competent packets (raw F/F0: 0.09 0.02 versus 0.40 0.06,

P < 0.01,

t-test).Th us, the capability of mobile vesicle clusters to

undergo destaining and a stimulus-evoked rise in Ca 2 + at fusion

sites are directly related.

Discussion

Using hippocampal neurons from mature cultures and acute slice, we have characterized key dynamic features of vesicle turnover along axons. Mobile fusion, in which discrete axonal regions behave as transiently active signalling sites contingent on the presence of traffi cking vesicles, is an important departure from the conventional model of point-to-point neurotransmission.

Our ndings are compatible with studies in other systems that have detailed processes of ectopic or extrasynaptic transmission 513. In particular, these examples have provided valuable information on how non-synaptic vesicle fusion can have a functional role in signalling to other neuronal 5 or non-neuronal targets 7,9,11. Nonetheless, insights into presynaptic processes that facilitate Ca 2+ -dependent release of vesicles at axonal sites away from conventional synaptic terminals have not been directly addressed. Our ndings here show that axonally derived signalling is a consequence of the fusion of traffi cking vesicles arising from the laterally mobile vesicle pool 1418,24,43,44. Th ese vesicles are typically

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

ARTICLE

FM1-43 PSD-95 (post)

FM1-43 GluR1

FM1-43 GluR2

s s

FM1-43 GABA-A-R16 0 6 12 16 s

PSD95 SytI

GluR1 GluR2

Figure 6 | Postsynaptic targets for mobile fusion. ( a) Sample panels from time-lapse sequences showing movement and destaining (40 Hz after 0 s) of mobile FM1-43-labelled packets (arrowheads). Bottom panels show post-x anti-PSD-95 with Alexa 568 immunostaining. Scale bars, 1 m. (b,c) Sample panels from dual-imaging time-lapse sequences showing movement and destaining (40 Hz after 0 s) of mobile FM1-43-labelled packets (arrowheads) and live antibody labelling for GluR1 or GluR2. Mobile packets move to sites adjacent to GluR targets and destain. Scale bars, 1m. (d) Sample panels from dual-imaging time-lapse sequence showing movement and destaining (40 Hz after 0 s) of mobile FM1-43-labelled packets (arrowheads) and live antibody labelling for GABA-A receptor. Scale bar, 1 m. (e) Sample kymographs showing the lack of lateral movement of GluR. Scale bar, 1 m. Vertical time bar, 10 s. ( f) Sample kymograph showing no evidence of lateral movement of ECFP-PSD-95 whereas the trafcking of vesicles labelled with SytI-Oyster550 along the apposed process is clearly evident. Scale bar, 2 m. Vertical time bar, 10 s.

stabilized by action potentials and subsequently undergo fast exocytosis. Notably, the onset of fusion is delayed, consistent with a period in which vesicles might be interacting with target proteins at axonal sites before exocytosis can occur; in support of this, we show that mobile vesicle packets that had spontaneously stabilized just before stimulation could undergo exocytosis immediately. Also, we noted examples of packets that delayed stimulus-evoked dye loss until they reached a GluR-apposed target region; we speculate that perhaps these packets do not immediately access molecular machinery required to support fusion. Using endocytic reporters, we demonstrate that vesicles involved in mobile fusion can be re-acidied and re-mobilized and subsequently undergo further fusion events. Th us, mobile vesicles, like those at conventional terminals 45, can be locally recycled to support ongoing signalling demands.

A confounding feature of regulated axonal fusion / recycling is that it runs against the established idea that Ca 2+ -dependent vesicle turnover is completely reliant on stably arranged, highly specialized presynaptic terminals. Our ndings oer some insight into this. We demonstrate that sites along the axon that are not associated with stable synaptic terminals can support reliable action-potential-dependent rises in intracellular Ca 2+ with kinetics comparable to those at conventional terminals. Such sites were readily distinguishable from typical axonal segments where stimulus-driven Ca 2+ changes were smaller and slower, as reported previously 41,42 and which presumably arise in large part from the lateral spread of Ca 2+ from nearby terminals. Ca 2+ hotspotswouldappear to provide the necessary large local rise in Ca 2+ to support mobile fusion of traffi cking vesicles. Consistent with our ndings, a recent work in corpus callosal neurons has demonstrated axonal release involv-

ing highly localized calcium microdomain signalling 7. Another issue is how is the necessary vesicle recycling machinery might be made available for turnover of vesicles in axonal regions? A previous work has demonstrated that packets of laterally mobile vesicles can label immunopositive for synaptic proteins 16, suggesting that some critical recycling machinery might already be present near fusion sites for local recruitment to traffi cking vesicles; for example, the target soluble NSF attachment proteins receptors are known to be homogeneously distributed along axonal membranes 46. How

ever, our ndings are not consistent with the idea that axonal release sites are small presynaptically silent synapses that become functionalized by the arrival of recycling vesicles because fusion events can readily occur at sites without a pre-existing vesicle pool. Alternatively, some synaptic components could themselves traffi c to axonal sites to support mobile release consistent with work showing that many synapse-related elements are highly laterally mobile 4753.

We demonstrate that actin is dynamically recruited to mobile vesicle packets. Interestingly, this recruitment occurs with some delay, suggesting that actin might not be critical for mobile fusion per se but acts in a supporting function, similar to its proposed role in stable terminals 35.

Th e fact that we observe mobile recycling in mature cultured neurons and native tissue suggests that this process is directly relevant to understanding signalling in established and native networks. Importantly, examples of mobile vesicle fusion have been very elegantly demonstrated in a variety of developing neuronal systems 2127 and this is thought to be an important step in nascent synapse formation 54.The specic relationship between this immature signalling process and the phenomenon we examine here is not established. Nonetheless, evidence suggests that, at a mechanistic

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

Time-lapse

Fix / PC

30 Hz

40 s

100

120 AP Stim

0 20

Release site

Dendrite

Axon

PC+

Figure 7 | Ultrastructure of axonal fusion sites. ( a) Schematic of experiment. Neurons were loaded with FM1-43FX and imaged during stimulation(120 AP, 30 Hz) after which they were xed, photoconverted (PC) and prepared for electron microscopy. ( b) Mean destaining traces showing that before xing, dye loss for 120 AP stimulation protocol (red) was equivalent to a sister culture where stimulation was maintained (blue). ( c) Left, sample time-lapse frames from a target region showing a mobile packet (white arrowhead) moving to new site and undergoing stimulation-evoked dye loss ina typically delayed manner compared with the adjacent stable synapse (blue arrowhead). Right, example of mobile packet (yellow arrowhead) merging with another small packet and undergoing destaining. Scale bar, 0.5 m. (d) Fluorescence image showing target site from ( c) with arrowheads marking the same structures. ( e) Electron micrograph of target region from ( d) with uorescence overlay illustrating correlative approach. ( f) Electron micrograph of same region from ( e) with dendrite marked in brown and mobile release sites indicated with dashed rectangles. Scale bars for ( df): 0.5m. (g) High-magnication electron micrograph of left target region (dashed rectangle) from ( f). (h) High-magnication electron micrograph of right target region (dashed rectangle) from ( f). Both ( g) and ( h) show vesicles with dark lumen corresponding to recycling vesicles, docked vesicles (white arrowheads) and specialized postsynaptic structures (black arrows). Scale bars for ( g) and ( h): 0.5m. (i) Reconstruction for release site in ( g). Scale bar, 0.25 m.

level, these two signalling processes are quite distinct using, for example, dierent molecular machinery for exocytosis and exhibiting dierent pharmacological sensitivity 16.

Our ultrastructural data indicates that mobile fusion sites can have characteristics of conventional terminals at the moment of fusion, including closely apposed postsynaptic targets, suggesting that they could have a direct role to play in information transmission. Over the time course of our experiments we did not see evidence to suggest substantial lateral movement of labelled aggregates of postsynaptic markers. Th is indicates that it is the traffi cking of presynaptic vesicles that largely accounts for the apposition of presynaptic markers with postsynaptic proteins observed soon aer stimulation. Of course, mobility in terms of single receptors is highly dynamic 55 and we cannot rule out the possibility of coordinated interactions occurring at the level of individual postsynaptic proteins. Importantly, mobile vesicle fusion onto postsynaptic targets would have very dierent signalling properties compared with conventional terminals. For example, their transmission capabilities should be directly dependent on vesicle traffi c: a parameter that appears to be specically regulated 17,56 . Moreover, functional consequences of extrasynaptic signalling might be very dierent: for example, activating distinct classes or combinations of target receptors compared with conventional terminals similar to the functional compartmentalization of signalling in chick ciliary ganglia 5 or perhaps even activate extrasynaptic receptor pools known to contribute to information transmission in hippocampal neurons 57.Th is has implications for plasticity 58 or signalling related to disease conditions 59,60.Th us, further knowledge about the mechanisms regulating synaptic vesicle traffi c and axonal fusion as well as the specic receptor targets involved in this process will provide new insights into important aspects of neuronal operation.

Methods

Cell culture and transfections. Dissociated hippocampal cultures were prepared from P0-P1 rats by plating neurons on an astrocyte feeder layer, and maintained

in BME media with 45 % glucose, 2 % FCS and B27 supplement. Mature hippocampal neurons were used for experiments a er 11 32 days in vitro. Experiments were performed in accordance with the UK-Animal (Scientic Procedures) Act 1986 and complied with local institutional regulations. Neurons were transfected at days in vitro 79, using a Ca 2+ phosphate protocol. Unless otherwise stated, all experiments were performed in external bath solution (137 mM NaCl, 5 mM KCl, 2.5mM CaCl2, 1mM MgCl2, 10mM d-Glucose, 5mM HEPES, 20M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 M d(-)-2-amino-5-phosphonovaleric acid (AP5)) at 25C.

Labelling and imaging of cultured neurons. Recycling synaptic vesicles were labelled using eld stimulation (600 APs, 10 Hz) in the presence of the dye (FM1-43 or its xable analogue FM1-43FX, FM4-64 , 10 M, Molecular Probes ). Aer loading, cells were washed with fresh external bath solution for 10 min to remove surface dye. In some experiments, vesicles were labelled using uorescently tagged antibodies directed to the vesicle protein synaptoptagmin I ( anti-synaptotagmin Oyster550, anti-synaptotagmin CypHer5E , 1:200, Synaptic Systems ) using the loading and washing protocol outlined above. Similarly, live labelling of postsynaptic structures ( anti-GluR1, Calbiochem; anti-GluR2 , Millipore , 1:200; anti-GABAA receptor 2,3 Chain , Millipore , 1:400) was carried out at 37 C for 30 min followed by secondary labelling (anti-rabbit or anti-mouse Alexa 488 or 568, 1:100). Plasmid constructs used were ECFP-PSD-95, SypHy2x, SypI-mCherryand GFP-actin. Imaging of living and xed cells was carried out with an Olympus

BX61WI microscope using a X60 1.0 N.A. dipping objective. Excitation and emission lter sets for uorescent probes were as follows: ECFP-PSD-95, 438 / 24, 470/22; FM1-43, SypHy2x and GFP-actin, 480/20, 520/35; FM4-64, 470/22, 624/40; SytI-Oyster550, 556/20, 609/54; SypI-mCherry 556/20, 624/40; CypHer5E, 628/40, 692 / 40. Image analysis was performed using ImageJ ( http://rsb.info.nih.gov/ij/ )on raw unltered images or aer ltering (1 1 median lter) applied to the whole image. For each mobile packet, we rst characterized the movement dynamics by generating a kymograph from a line prole that traced the axon. The kymograph was then used as a guide to dene the region of interest (ROI) for our packetin the time-lapse image sequence. To extract raw uorescence intensity valuesfor packets, we targeted the packet in the frame before stimulation. An ROI was drawn that encapsulated the packet and anking axon (~1m on each side). In the subset of cases where packets continued to move a er stimulation, multiple ROIs were used to track uorescence intensity along the axon, using the kymographas a guide. Background uorescence was subtracted from an ROI adjacent to the target packet but that did not include the axon or other structures. For analysis of mobility rates in relation to stimulation, we generated kymographs and overlaid linear proles that described the lateral movement over time. Movement rates before and a er stimulation could be extracted by measuring the angles of the line

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

ARTICLE

SypI-mCherry OGB-1

SypI-mCherry

Rise (ms)

1.2

0.8

0.4

Peak

Decay (ms)

600

400

200

0 S H

A S H

AP AP AP AP

OGB-1 FM4-64

120

960

PSD95

Time

0 100%F/F

Figure 8 | Action potential dependent Ca2+ entry at axonal sites. ( a) Image overlay showing a SypI-mCherry-expressing neuron (red) lled with OGB-1 (green). The pipette is indicated with white lines. Scale bar, 10 m. (b) Four single-action potential-evoked Ca 2+ responses at a synapse (blue box) and the anking axonal region (grey box) marked in uorescence image (top). Scale bar, 1 m. Timescale bar 1 s. Vertical scale bar: 20 % F/F. (c) Time-lapse sequence showing movement of SypI-mCherry packets (green arrowheads) along axonal region next to stable synapse (blue arrowhead). Scale bar, 1 m. ( d) Sample Ca 2+ response traces from ( c). Blue trace is stable synapse, grey trace is axonal region and green trace is Ca 2+ hotspot (e). Timescale bar 100 ms. Vertical scale bar: 50 % F/F. (e) Line scan (yellow line in c) showing Ca 2+ responses to four single-action potentials. Blue arrowhead, stable synapse; grey arrowhead, axonal region; green arrowheads, Ca 2+ hotspot. Scale bar, 1m. Timescale bar, 1 s. ( f) Histograms showing mean s.e.m. for 3 parameters of Ca 2+ response ( n = 4 from 4 / 2, with each sample averaged from 4 action potential-evoked responses) for synapse (S), axon (A) and hotspot (H). For clarity, normalized F/F0 values are shown in histogram. Raw F/F0 were synapse, 0.58 0.13; hotspot: 0.570.16; and axon:0.090.05 (n=4, each averaged from 4 responses). (g) Sample of experiment monitoring action potential-evoked Ca 2+ responses and FM-64 destaining sequentially. Time-lapse frames on left show rise in OGB-1-AM uorescence evoked by single-action potential. Yellow arrowhead indicates hotspot . (right) Time-lapse frames immediately following Ca 2+ imaging showing movement and dye loss of mobile packet (arrowhead) at the same site with 40 Hz stimulation. Bottom right panel shows post-x immunolabelling of anti-PSD-95 with Alexa 568 indicating apposition to postsynaptic target at destaining site. Scale bar 1 m.

prole and converting to distance and time units. Quantication of the occurrence of mobile-fusion events relative to dye-loss events at stable synapses was carried out by analysis of kymographs generated from line proles along discrete axonal segments. Segments ( n=50) were chosen that had a dened stable terminal and a anking axonal length (3.5 m) that did not include other stable terminals. Mobile fusion events were only counted if they were mobile before the stimulus and could be distinguished from dye loss at the stable synapse. Curve tting was carried out with Prism or Igor. Statistical comparisons used appropriate two-tailed t-tests unless otherwise stated. Signicance was dened as P<0.05.

Acute slice preparation. Acute transverse slice preparations of hippocampus (300m) were prepared from 18 24 d rats. Presynaptic terminals were labelled using a protocol similar to those described previously 30,31. Briey, slices were loaded with bath application of FM1-43 (20 M) in aCSF containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 1.25 NaH 2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2 and 50M AP5 (pH 7.3 when bubbled with 95 % O2 and 5 % CO2). Slices were incubated in FM1-43 solution (3 min) and subsequently a 1,200 AP 10 Hz loading stimulation was delivered using a bipolar tungsten electrode placed near Schaer collaterals. Two minutes a er the end of stimulation slices were washed in continuously perfused aCSF (10 min) and then in the FM-dye chelator ADVASEP-7 (1 mM, Biotium , 1min). Finally, slices were continuously perfused with aCSF for>20min. Before FM-dye loading the target CA1 region was chosen on the basis of the electrical

response to stimulation recorded with an aCSF-lled glass electrode (23M); to allow postsynaptic responses to be recorded, AMPARs were not blocked in these experiments. Imaging of FM-dye-labelled presynaptic terminals used an Olympus BX51WI microscope equipped with a FV-300 confocal system ( Olympus UK ),a 488-nm Ar laser and 520 / 10 emission. Confocal time-lapse image sequences (optical section thickness<1.4m) were acquired with 2-second intervals between frames; the rate required to track movements of packets.

Immunouorescence. Hippocampal cultures were xed with either 4 % paraformaldehyde in PBS or 4 % paraformaldehyde and 1 % glutaraldehyde in PBS. The preparations were washed and quenched in 50 mM NH 4Cl and permeabilized in 0.2 % Triton X-100 PBS. A er blocking with goat serum, cells were incubated in anti-mouse glutamate (ImmunoStar, 1:2,000) or anti-PSD-95 (Abcam, 1:200), followed by a secondary incubation with goat anti-mouse Alexa 488 or 568 ( Invitrogen , 1:500). Coverslips were mounted in uorescence mounting medium (DAKO) for imaging.

Photoconversion and electron microscopy. Hippocampal cultures labelled with FM1-43FX were xed and photoconverted using a previously established protocol 15,39. Briey, neurons were xed in 2% PFA/2% glutaraldehyde for 15min, washed with 100 mM glycine (1 h) followed by NH 4Cl (1 min) and rinsed in PBS.

Photoconversion was obtained by illuminating the samples with 475 / 40 nm light

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

from a mercury lamp for 12 min in the presence of diaminobenzidine ( DAB , 1mgml 1, DAKO ). Following photoconversion, samples were osmicated, stained and embedded in EPON ( TAAB ) 15,39. Embedded samples were sectioned and collected on formvar-coated slot grids. Sections were viewed using a Hitachi-7100 transmission electron microscope and images acquired using a 2,0482,048 CCD camera ( Gatan ). Micrographs were aligned and reconstructed using Xara Xtreme and Reconstruct ( Synapse Web , Kristen M. Harris, http://synapses.clm.utexas.edu/ ).

Electrophysiology and calcium imaging. Whole-cell recordings were obtained using patch pipettes (3 5 M resistance) lled with 115 mM potassium methylsulphate, 5 mM potassium chloride, 10 mM HEPES buer, 10 mM creatine phosphate, 2mM ATP-Mg, 2mM ATP-Na 2, 0.3mM. GTP-Na2 and 200M Oregon Green 488

BAPTA-1 (OGB-1) or Fluo-5F (Molecular Probes). Voltage recordings were carried out using a Multiclamp 700A amplier and digitized using a Digidata 1320a (Molecular Devices). Electrophysiological traces were visualized and analysed using pClamp so ware ( Molecular Devices ). Calcium imaging experiments were carried out using an Olympus BX51WI microscope equipped with a FV-300 confocal system ( Olympus UK ). SypI-mCherry and OGB-1 or Fluo-5F were excited with a 543 nm HeNe laser and a 488 nm Ar laser ( CVI Melles-Groit ), respectively. Line scans were obtained at 500 750 Hz using a X60 water immersion objective (1.0 N.A., Olympus UK ). Th e relative changes in uorescence of OGB-1 or Fluo-5F were quantied as F/F0(t)=(F(t)F0)/F0, where F0 is the basal uorescence calculated averaging the intensity values of 100 ms preceding the action potential for each pixel in the line; % F/F0 was expressed as a percentage of the uorescence at the peak of the calcium signal at the synapse. Calcium signals were analysed using Matlab ( Th e Mathworks); peak amplitudes, 1090% rise and 100 % decay times were measured using Igor pro ( Wavemetrics ) with the Neuromatic package ( www.neuromatic.thinkrandom.com/ ). In experiments to characterize Ca 2+ hotspots and mobile fusion at the same sites, we loaded neurons with the membrane permeable form of OGB-1 (OGB-1-AM, 200 M, Molecular Probes, 2 h incubation at 37 C). Synapses were labelled with FM4-64 (600 APs, 10 Hz) as above. Images of OGB-1-AM signal were collected at 50 Hz for 4 s usinga CCD system (above) and a single AP was evoked by eld stimulation. Time-lapse imaging for FM-dye signal (every 2 s) was initiated immediately a er this and the culture destained at 40Hz.

References

1. Sudhof, T. C. Th e synaptic vesicle cycle . Annu. Rev. Neurosci. 27, 509547 (2004).

2. Matsui, K. & Jahr, C. E. Exocytosis unbound.Curr. Opin. Neurobiol. 16,

305311 (2006).

3. Szapiro, G. & Barbour, B. Parasynaptic signalling by fast neurotransmitters: the cerebellar cortex.Neuroscience 162, 644655 (2009).

4. Kullmann, D. M. Spillover and synaptic cross talk mediated by glutamate and GABA in the mammalian brain . Prog. Brain Res. 125, 339351 (2000).

5. Coggan, J. S. et al. Evidence for ectopic neurotransmission at a neuronal synapse.Science 309, 446451 (2005).

6. Khimich, D.et al. Hair cell synaptic ribbons are essential for synchronous auditory signalling.Nature 434, 889894 (2005).

7. Kukley, M., Capetillo-Zarate, E. & Dietrich, D. Vesicular glutamate release from axons in white matter . Nat. Neurosci. 10, 311320 (2007).

8. Lenzi, D., Crum, J., Ellisman, M. H. & Roberts, W. M. Depolarization redistributes synaptic membrane and creates a gradient of vesicles on the synaptic body at a ribbon synapse . Neuron 36, 649659 (2002).

9. Matsui, K. & Jahr, C. E. Ectopic release of synaptic vesicles.Neuron 40, 11731183 (2003).

10. Zenisek, D., Davila, V., Wan, L. & Almers, W. Imaging calcium entry sites and ribbon structures in two presynaptic cells . J. Neurosci. 23, 25382548 (2003).

11. Ziskin, J. L., Nishiyama, A., Rubio, M., Fukaya, M. & Bergles, D. E. Vesicular release of glutamate from unmyelinated axons in white matter . Nat. Neurosci. 10, 321330 (2007).

12.Beutner, D., Voets, T., Neher, E. & Moser, T. Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell aerent synapse.Neuron 29, 681690 (2001).

13.Fields, R. D. & Ni, Y. Nonsynaptic communication through ATP release from volume-activated anion channels in axons . Sci. Signal. 3, ra73 (2010).

14.Chen, X., Barg, S. & Almers, W. Release of the styryl dyes from single synaptic vesicles in hippocampal neurons . J. Neurosci. 28, 18941903 (2008).

15. Darcy, K. J., Staras, K., Collinson, L. M. & Goda, Y. Constitutive sharing of recycling synaptic vesicles between presynaptic boutons . Nat. Neurosci. 9, 315321 (2006).

16.Krueger, S. R., Kolar, A. & Fitzsimonds, R. M. Th e presynaptic release apparatus is functional in the absence of dendritic contact and highly mobile within isolated axons.Neuron 40, 945957 (2003).

17. Staras, K. et al. A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons.Neuron 66, 3744 (2010).

18. Westphal, V. et al. Video-rate far-eld optical nanoscopy dissects synaptic vesicle movement.Science 320, 246249 (2008).

19.Staras, K. & Branco, T. Sharing vesicles between central presynaptic terminals: implications for synaptic function . Front. Synapt. Neurosci. 2, 20 (2010).

20.Staras, K. Share and share alike: trading of presynaptic elements between central synapses.Trends Neurosci. 30, 292298 (2007).

21. Matteoli, M., Takei, K., Perin, M. S., Sudhof, T. C. & De Camilli, P. Exoendocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.J. Cell Biol. 117, 849861 (1992).

22. Antonov, I., Chang, S., Zakharenko, S. & Popov, S. V. Distribution of neurotransmitter secretion in growing axons . Neuroscience 90, 975984 (1999).

23.Dai, Z. & Peng, H. B. Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis . Mol. Cell. Neurosci. 7, 443452 (1996).

24. Hopf, F. W., Waters, J., Mehta, S. & Smith, S. J. Stability and plasticity of developing synapses in hippocampal neuronal cultures . J. Neurosci. 22, 775781 (2002).

25. Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin . J. Neurosci. 15, 43284342 (1995).

26.Verderio, C., Coco, S., Pravettoni, E., Bacci, A. & Matteoli, M. Synaptogenesis in hippocampal cultures.Cell. Mol. Life Sci. 55, 14481462 (1999).

27. Zakharenko, S., Chang, S., ODonoghue, M. & Popov, S. V. Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis.J. Cell Biol. 144, 507518 (1999).

28.Betz, W. J. & Bewick, G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction . Science 255, 200203 (1992).

29. Ryan, T. A. et al. Th e kinetics of synaptic vesicle recycling measured at single presynaptic boutons.Neuron 11, 713724 (1993).

30.Unni, V. K., Zakharenko, S. S., Zablow, L., DeCostanzo, A. J. & Siegelbaum, S.A. Calcium release from presynaptic ryanodine-sensitive stores is required for long-term depression at hippocampal CA3-CA3 pyramidal neuron synapses . J. Neurosci. 24, 96129622 (2004).31.Zakharenko, S. S., Zablow, L. & Siegelbaum, S. A. Visualization of changes in presynaptic function during long-term synaptic plasticity . Nat. Neurosci. 4, 711717 (2001).

32. Welzel, O. et al. Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses . Biophys. J. 100, 593601 (2011).

33. Granseth, B., Odermatt, B., Royle, S. J. & Lagnado, L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.Neuron 51, 773786 (2006).

34.Colicos, M. A., Collins, B. E., Sailor, M. J. & Goda, Y.Remodeling of synaptic actin induced by photoconductive stimulation . Cell 107, 605616 (2001).

35. Sankaranarayanan, S., Atluri, P. P. & Ryan, T. A. Actin has a molecular scaolding, not propulsive, role in presynaptic function . Nat. Neurosci. 6, 127135 (2003).

36. Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J. & Tsien, R. W. Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion.Proc. Natl Acad. Sci. USA 98, 1274812753 (2001).

37.Rizzoli, S. O. & Betz, W. J. Th e structural organization of the readily releasable pool of synaptic vesicles . Science 303, 20372039 (2004).

38.Schikorski, T. & Stevens, C. F. Morphological correlates of functionally dened synaptic vesicle populations.Nat. Neurosci. 4, 391395 (2001).

39. Darcy, K. J., Staras, K., Collinson, L. M. & Goda, Y. An ultrastructural readout of uorescence recovery a er photobleaching using correlative light and electron microscopy.Nat. Protoc. 1, 988994 (2006).

40.Zucker, R. S. Exocytosis: a molecular and physiological perspective.Neuron 17, 10491055 (1996).

41.Koester, H. J. & Sakmann, B. Calcium dynamics associated with action potentials in single nerve terminals of pyramidal cells in layer 2/3 of the young rat neocortex.J. Physiol. 529 (Pt 3), 625646 (2000).

42.Mackenzie, P. J., Umemiya, M. & Murphy, T. H. Ca2+ imaging of CNS axons in culture indicates reliable coupling between single action potentials and distal functional release sites.Neuron 16, 783795 (1996).

43.Fernandez-Alfonso, T. & Ryan, T. A. A heterogeneous resting pool of synaptic vesicles that is dynamically interchanged across boutons in mammalian CNS synapses.Brain Cell Biol. 36, 87100 (2008).

44. Zhang, Q., Li, Y. & Tsien, R. W. Th e dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles . Science 323, 14481453 (2009).

45. Fernandez-Alfonso, T. & Ryan, T. A. The effi ciency of the synaptic vesicle cycle at central nervous system synapses . Trends Cell Biol. 16, 413420 (2006).

46. Garcia, E. P., McPherson, P. S., Chilcote, T. J., Takei, K. & De Camilli, P.

rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin . J. Cell Biol. 129, 105120 (1995).47. Frischknecht, R., Fejtova, A., Viesti, M., Stephan, A. & Sonderegger, P. Activity-induced synaptic capture and exocytosis of the neuronal serine protease neurotrypsin.J. Neurosci. 28, 15681579 (2008).

48. Kalla, S. et al. Molecular dynamics of a presynaptic active zone protein studied in Munc13-1-enhanced yellow uorescent protein knock-in mutant mice . J. Neurosci. 26, 1305413066 (2006).49.Li, Z. & Murthy, V. N. Visualizing postendocytic traffi c of synaptic vesicles at hippocampal synapses.Neuron 31, 593605 (2001).

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1534

ARTICLE

50. Matz, J., Gilyan, A., Kolar, A., McCarvill, T. & Krueger, S. R. Rapid structural alterations of the active zone lead to sustained changes in neurotransmitter release.Proc. Natl Acad. Sci. USA 107, 88368841 (2010).

51. Star, E. N., Newton, A. J. & Murthy, V. N. Real-time imaging of Rab3a and Rab5a reveals dierential roles in presynaptic function . J. Physiol. 569, 103117 (2005).

52. Tsuriel, S. et al. Exchange and redistribution dynamics of the cytoskeleton of the active zone molecule bassoon . J. Neurosci. 29, 351358 (2009).

53. Tsuriel, S. et al. Local sharing as a predominant determinant of synaptic matrix molecular dynamics.PLoS Biol. 4, e271 (2006).

54. Matteoli, M., Coco, S., Schenk, U. & Verderio, C. Vesicle turnover in developing neurons: how to build a presynaptic terminal . Trends Cell Biol. 14, 133140 (2004).

55.Choquet, D. Fast AMPAR traffi cking for a high-frequency synaptic transmission.Eur. J. Neurosci. 32, 250260 (2010).

56. Bamji, S. X., Rico, B., Kimes, N. & Reichardt, L. F. BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-beta-catenin interactions.J. Cell Biol. 174, 289299 (2006).

57. Okubo, Y. et al. Imaging extrasynaptic glutamate dynamics in the brain. Proc. Natl Acad. Sci. USA 107, 65266531 (2010).

58.Kullmann, D. M. & Asztely, F. Extrasynaptic glutamate spillover in the hippocampus: evidence and implications . Trends Neurosci. 21, 814 (1998).

59. Milnerwood, A. J. et al. Early increase in extrasynaptic NMDA receptor signaling and expression contributes to phenotype onset in Huntingtons disease mice.Neuron 65, 178190 (2010).

60. Okamoto, S. et al. Balance between synaptic versus extrasynaptic NMDA receptor activity inuences inclusions and neurotoxicity of mutant huntingtin . Nat. Med. 15, 14071413 (2009).

Acknowledgements

We gratefully acknowledge R.F. Toonen (Vrije Universitat, NL) for gi s of the Syphy2x and SypI-mCherry constructs and Y. Goda (UCL, UK) for GFP-actin. Research supported by Wellcome Trust (WT084357MF) and BBSRC (BB / F018371) grantsto K.S.

Author contributions

A.R., V.M. and K.S. performed the experiments, analysed the data and prepared the gures. Experiments were conceived by K.S. and designed with A.R., V.M. and T.B. Th e manuscript was written by K.S. with contributions from A.R., V.M. and T.B.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications

Competing nancial interests: Th e authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/

How to cite this article: Ratnayaka, A. et al. Extrasynaptic vesicle recycling in mature hippocampal neurons. Nat. Commun. 2:531 doi: 10.1038 / ncomms1534 (2011).

License: Th is work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-sa/3.0/

NATURE COMMUNICATIONS | 2:531 | DOI: 10.1038/ncomms1534 | www.nature.com/naturecommunications

Word count: 9981

Show less

Abstract

Translate

Fast neuronal signalling relies on highly regulated vesicle fusion and recycling at specialized presynaptic terminals. Recently, examples of non-classical neurotransmission have also been reported, where fusion of vesicles can occur at sites remote from conventional synapses. This has potentially broad biological implications, but the underlying mechanisms are not well established. Here we show that a complete vesicle recycling pathway can occur at discrete axonal sites in mature hippocampal neurons and that extrasynaptic fusion is a robust feature of native tissue. We demonstrate that laterally mobile vesicle clusters trafficking between synaptic terminals become transiently stabilized by evoked action potentials and undergo complete but delayed Ca²⁺ -dependent fusion along axons. This fusion is associated with dynamic actin accumulation and, subsequently, vesicles can be locally recycled, re-acidified and re-used. Immunofluorescence and ultrastructural work demonstrates that extrasynaptic fusion sites can have apposed postsynaptic specializations, suggesting that mobile vesicle recycling may underlie highly dynamic neuron-neuron communication.

Details

Title

Extrasynaptic vesicle recycling in mature hippocampal neurons

Author

Ratnayaka, Arjuna; Marra, Vincenzo; Branco, Tiago; Staras, Kevin

Pages

531

Publication year

2011

Publication date

Nov 2011

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms1534

ProQuest document ID

906644170

Extrasynaptic vesicle recycling in mature hippocampal neurons

Jump to:

Full text

Abstract

Details

Suggested sources