Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y

Accepted Manuscript Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y Anneka Bost, Ali H. Shaib, Yvonne Schwarz, Barbara A. Niemeyer, Ute Becherer PII: DOI: Reference:

S0306-4522(17)30012-X http://dx.doi.org/10.1016/j.neuroscience.2017.01.006 NSC 17542

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

15 March 2016 3 January 2017 4 January 2017

Please cite this article as: A. Bost, A.H. Shaib, Y. Schwarz, B.A. Niemeyer, U. Becherer, Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.01.006

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Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y

Anneka Bost1, Ali H. Shaib 1,3, Yvonne Schwarz1, Barbara A. Niemeyer2, Ute Becherer1* 1

Institute of Physiology, CIPMM, Saarland University, 66421 Homburg/Saar, Germany

2

Institute of Biophysics, CIPMM, Saarland University, 66421 Homburg/Saar, Germany

3

Present address: Department of Molecular Neurobiology, Max Planck Institute of

Experimental Medicine, 37075 Göttingen, Germany

Running title: LDCV release in DRG neurons

Corresponding author *: Ute Becherer Institute of Physiology CIPMM Saarland University D-66421 Homburg/Saar, Germany Tel.: +49-(0)6841-16-16409 Fax: +49-(0)6841-16-16402 E-mail: [email protected]

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Abbreviations: large dense-core vesicle (LDCV), neuropeptide Y (NPY), calcitonin-gene related peptide (CGRP), dorsal root ganglion (DRG), total internal reflection fluorescence microscopy (TIRFM), adenylate cyclase (AC), L-type voltage dependent Ca2+ channels (VDCC), brain derived neurotrophic factor (BDNF)

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Abstract Peptidergic dorsal root ganglion (DRG) neurons transmit sensory and nociceptive information from the periphery to the central nervous system. Their synaptic activity is profoundly affected by neuromodulatory peptides stored and released from large densecore vesicles (LDCVs). However, the mechanism of peptide secretion from DRG neurons is poorly understood. Using total internal reflection fluorescence microscopy (TIRFM), we visualized individual LDCVs loaded with fluorescent neuropeptide Y (NPY) and analyzed their stimulation-dependent release. We tested several protocols and found an overall low stimulation-secretion coupling that increased after raising intracellular Ca2+ concentration by applying a weak pre-stimulus. Interestingly, the stimulation protocol also influenced the mechanism of LDCV fusion. Depolarization of DRG neurons with a solution containing 60 mM KCl triggered full fusion, kiss-and-run, and kiss-and-stay exocytosis with equal frequency. In contrast, field electrode stimulation primarily induced full fusion exocytosis. Finally, our results indicate that NPY can promote LDCV secretion. These results shed new light on the mechanism of NPY action during modulation of DRG neuron activity, an important pathway in the treatment of chronic pain.

Keywords: dorsal root ganglion neuron, large dense-core vesicles, exocytosis, neuropeptide Y, TIRF-microscopy, kiss-and-run.

Introduction Neurons convey information not only through synaptic transmission, but also via release of large dense-core vesicle (LDCV) content that is extremely diverse. In most neurons they are filled with neurotrophins, but they can also contain hormones or neuromodulatory peptides. LDCVs are localized in the cell body as well as at synapses. While their exocytosis has been studied in detail in endocrine and neuroendocrine cells, little is known about their fusion in neurons. In hippocampal neurons LDCVs are mainly loaded with growth factors and their exocytosis is induced by strong and long lasting stimuli indicating relatively weak stimulation-secretion coupling (Xia et al., 2009). Furthermore, most LDCV exocytosis appears to occur by kiss-and-run fusion events (Lessmann and Brigadski, 2009). In comparison, in 3

neuroendocrine cells LDCVs are filled with hormones and accumulate in readily releasable pools (Sorensen, 2004, Becherer and Rettig, 2006). They are released mainly through full fusion exocytosis, while kiss-and-run exocytosis only occurs upon very weak stimulation (Fulop et al., 2005). We set out to investigate whether these differences are due to the function of different LDCV contents or whether there are fundamental differences in the LDCV release mechanisms between neuroendocrine cells and neurons. To address these questions, we selected neurons that use mainly synaptic transmission for information transfer, but also release LDCVs containing neuropeptides, which show rapid modulation of neuronal activity. Dorsal root ganglion (DRG) neurons meet these criteria as they convey sensory information to the central nervous system via excitatory glutamatergic transmission. A subset, called peptidergic neurons, contain a large amount of LDCVs filled with a variety of neuropeptides such as calcitonin-gene related peptide (CGRP), substance P (SP) or neuropeptide Y (NPY) (Hokfelt et al., 1980, Schoenen et al., 1989). The functions of these neuromodulatory peptides are quite diverse, but they can rapidly alter the excitability of neurons (Murase and Randic, 1984, Ryu et al., 1988, Randic et al., 1990) and thereby participate in chronic pain generation (for review see Pezet and McMahon (2006)). Although these functions of neuropeptides have been studied in detail, little is known about their mechanism of release. Studies using membrane capacitance recordings on isolated DRG neurons showed that LDCV secretion could be both Ca2+-dependent (Huang and Neher, 1996) but also Ca2+-independent (Zhang and Zhou, 2002, Zheng et al., 2009). Hence, understanding the stimulation-secretion coupling and the mechanism of LDCV release is of major interest. Here we studied the exocytosis of fluorescently stained LDCVs at the soma of DRG neurons with total internal reflection fluorescence microscopy (TIRFM). Various secretagogues and stimulation protocols were used to investigate stimulation-secretion coupling and mode of exocytosis. We found that 100 Hz field electrode stimulation preceded by 5 Hz stimulation resulted in reliable secretion. Furthermore, the type of LDCV fusion depended on the stimulation. While all fusion types occurred to a similar degree when DRG neurons were depolarized with a solution containing 60 mM KCl, field electrode stimulation induced primarily full fusion exocytosis. Finally, we showed that NPY can promote LDCV secretion.

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Experimental procedures DRG neuron preparation The protocol was adapted from Murakami et al. (2002). In brief, DRGs were dissected from 2-4 week old, B6/N mice or B6.FVB-Tg(Npy-hrGFP)1Lowl/J mice (The Jackson Laboratory, Bar Harbor, Maine, USA) and digested in 2.31 U Liberase DH (Roche, Mannheim, Deutschland) in NBA-medium (Gibco Life Technologies, Darmstadt, Germany) at 37°C for about 17 min. Ganglia were then mechanically triturated with a 1 ml Eppendorf pipette and cells were centrifuged at 300 rcf for 3 min. The supernatant was discarded and the cells were washed with 1 ml DPBS (Gibco). After a second centrifugation, cells were resuspended in NBA medium containing 2% B27 supplement (Invitrogen, Darmstadt, Germany), 1% Glutamax (Invitrogen) and 0.2% penicillin/streptomycin (both Invitrogen) and seeded on Poly-(D)-Lysine (Sigma-Aldrich, Steinheim, Germany) coated 25 mM glass coverslips. They were kept in medium containing additionally 5% FCS (Invitrogen) and 10 µl/ml FUDR (8.1 mM FUDR (Sigma-Aldrich), 20.5 mM Uridine (Sigma-Aldrich) in DMEM (Invitrogen)), to reduce non-neuronal cell growth. 24 H after isolation cells were transfected via lentivirus (see below). Cells were maintained 7 to 8 days in culture before performing the experiments. All experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany and the State of Saarland. Transfection To visualize LDCV secretion, DRG neurons were transfected with a lentivirus (pRRL.sin.cPPT.CMV.WPRE lentiviral transfer vector) encoding for the fusion protein NPYVenus, whereby NPY is specifically targeted to LDCVs in neurons (Ramamoorthy et al., 2011, van de Bospoort et al., 2012, Farina et al., 2015) and Venus is a pH dependent yellow fluorescent protein with a pKa of 6 (Nagai et al., 2002). The plasmid was generated by introducing a NPY-Venus PCR product (forward primer: 5’-TAT ATA GGT CTC GGA TCC ACC ATG CTA GGT AAC AAG-3’ purchased from Invitrogen; reverse primer: 5’-TAT AGC TAG CTT ACT TGT ACA GCT CGT CCA TG-3’ from AGCTLab, Göttingen, Germany) into an empty lentiviral vector via BsaI/BamHI and NheI restriction sites. The virus was produced using the ViraPowerTM Lentiviral Expression System from Invitrogen (K4975-00) (see Guzman et al. (2010)). Cells were infected after one day in vitro (DIV) in NBA-medium without FUDR and 5

penicillin/streptomycin. At DIV 2 the medium was changed back to medium with FUDR and penicillin/streptomycin. The lentivirus does not target a specific subset of DRG neurons. Thus peptidergic as well as non-peptidergic cells are transfected by the construct and will show similar punctate staining. Furthermore, non-neural cells such as astrocytes were also transfected by the virus albeit with much lower probability. In that case the entire field of view containing a transfected astrocyte was avoided (Fig. 1). Cells were used for experiments on day 7 or 8 in vitro to allow sufficient time for NPY-Venus expression and a large number of labeled LDCVs to be produced. Transfection efficiency was about 50 to 60%. TIRF Microscopy and Stimulation Acquisition was done at an inverted TIRF microscope (Olympus IX 70, Olympus, Hamburg, Germany) connected to a QuantEM 512SC (Photometrics, Tucson, AZ, USA) CCD camera, an 100x oil-immersion objective and the help of Metamorph software (Molecular Devices, LLC Sunnyvale, Ca, USA) (see also Becherer et al. (2007)). Movies were acquired at a rate of 10 Hz in stream modus. To illuminate the cells an Argon laser (450, 488, 514 nm) from Spectra-Physics (Darmstadt, Germany) in combination with an AOTF (A-A Opto-Electronic, Wallenhorst, Germany) was used. The filters were from AHF-Analysetechnik AG (Tübingen, Germany). The penetration depth of the evanescent wave was about 230 nm (Quintana et al., 2009). DRG neurons were superfused with an extracellular solution containing (in mM) 152 NaCl, 2.4 KCl, 10 HEPES, 1.2 MgCl2, 2.5 CaCl2, 10 Glucose (pH 7.4). Experiments were performed at 32°C (Eight Line In-Line Solution Heater (SHM-828) and CL-100 Bipolar Temperature Controller, Warner Instruments, Hamden, CT, USA). DRG neurons in vivo and in vitro do not form autaptic synaptic connection and they are electrically silent (Ransom et al., 1977a, Ransom et al., 1977b). They fire single action potential upon brief field electrode stimulation, which allows precise regulation of pattern and firing frequency of neurons in cell culture (Ransom et al., 1977a). To induce LDCV secretion either a solution containing high [K+] was applied, in which 57.6 mM NaCl of the extracellular solution was replaced by KCl, or the cells were electrically stimulated with a field electrode (Isolated Pulse Stimulator Model 2100, AM Systems, Sequim, WA, USA). The amplitude of the electrical stimulation was 10 µA with a variety of pulse durations and frequencies. For NPY treatment, cells were bathed in

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extracellular solution containing 50 nM NPY (peptide # ab32971 from Abcam, Cambridge, UK) for 5 min. Secretion recorded in TIRFM movies was evaluated in the soma of the neurons manually with Image J (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014). To analyze the fusion mode, movies were background subtracted, and examined by eye to detect vesicles as round spots of about 3 to 5 pixel square that displayed sharp fluctuation of fluorescence intensity. The average fluorescence intensity of this spot was then measured in a time window of 3 s before and after the event using ROI and multimeasure functions of ImageJ. Additionally, the fluorescence intensity of its immediate surrounding was measured as well. Both were plotted in IgorPro (WaveMetrics, Lake Oswego, OR, USA). A customized IgorPro routine was used to automatically classify vesicles depending on their fusion modes (Fig. 2A). The first selection criteria was used to differentiate between a vesicle that lingers in the back of the evanescent field moving in and out of it and a vesicle at the plasma membrane that is secreted. For that, at the time of exocytosis, the fluorescent intensity of the selected spot should be at its own maximum and at least 4 times higher than background, before decreasing abruptly (in 200 ms). If these criteria were not met then the vesicle was not fusing with the plasma membrane and the spot was discarded. If they were met then the vesicles fluorescence upon fusion was compared to the local background. If the fluorescence intensity reached background level, within another 100 ms, then this event was likely to be a full fusion event. To confirm this we also analyzed the history of the vesicle. If the fluorescence intensity increase was slow or not existent (in the measured time window of 3 s) then the event was classified as full fusion. If instead its fluorescent intensity prior

exocytosis increased suddenly in less than 325 ms by more than 3 standard deviation (SD) of its own fluorescent intensity fluctuation then the event was categorized as kiss-and-run or kiss-and-stay. The rationale behind this selection criterion was that a very sharp increase in fluorescence corresponds to pH dequenching of Venus fluorescence upon fusion pore opening. A slower increase on the other hand, corresponded to a vesicle traveling through increasing light intensity of the evanescent wave as it approached the plasma membrane during docking, ie tethering. The selection criterion of 325 ms was chosen after generating a histogram with the fluorescent rise time of all vesicles, which was best fit by a sum of two lognormal (Fig. 2B). Their intersection point was 325 ms. In few cases a slow rise of 7

fluorescence was followed by a small fluorescence spike indicative of pH unquenching. These events were indistinguishable by our selection criteria form events in which only a slow increase of fluorescence occurred. Because the unquenching spikes were small (about 2 to 3 SD of fluorescence variation) we classified these events as full fusion. To distinguish between kiss-and-run and kiss-and-stay, we applied an additional criterion, in which we compared the fluorescence intensity of the spot 2 s before and after fusion of the vesicle with the plasma membrane. If the vesicle fluorescence intensity 2 s after fusion was reduced by more than 4 SDs of its intensity fluctuation in comparison to the fluorescence 2 s before fusion, then the fusion event was classified as kiss-and-run. Else the event was a kiss-and-stay event. Calcium Imaging For calcium imaging DRG neurons were loaded with 2 µM Fura-2 AM (Life Technologies) in Opti-MEM (Invitrogen) enriched with 0.2% bovine serum albumin (Sigma-Aldrich) at 37°C for 15 min. The dye was excited with UV-light alternating between 350 and 380 nm using a monochromator (Visitron, Puchheim, Germany) and movies were acquired in Metamorph at a rate of 10 Hz. Here a 40× oil-immersion objective was used. For the analysis ROIs marking cell soma were generated in ImageJ and the average gray values were transferred to Excel. IB4 staining and Immunocytochemistry Isolectin GS-IB 4 staining was performed on live cells. IB 4 is used to mark the non-peptidergic unmyelinated DRG neurons (Kubo et al., 2012). DRG neurons were labeled with 0.25 µg/ml IB4-Alexa 568 (Invitrogen) in extra-cellular solution and incubated at 37 °C for 20 min. Cells were then washed for at least 10 min with continuous extracellular solution perfusion. For immunocytochemistry, cells were fixed with 4% paraformaldehyde in PBS-NaCl followed by a 50 mM glycine block and a permeabilization step with 0.1% TritonX and 2.5% NGS. The antibodies against NPYR1 and NPYR2 (rabbit antibody ANR-021 and ANR-022 respectively; Alomone, Jerusalem, Israel) were both used at a dilutions of 1:100. Secondary antibody was Alexa 568 anti-rabbit (# A11008, Invitrogen) at a dilution of 1:1000. Pictures were taken at a Zeiss LSM 780 System with ZEN software (Zeiss, Jena, Germany). Statistics Average values were calculated across all recorded cells that were originating from a minimum of 3 cultures. Statistical analysis was done in SigmaPlot 11.0. To compare two 8

groups of data, we used a Student t-test or a Mann-Whitney Rank Sum Test when the data was not following a normal distribution. If applicable a Two Way ANOVA was performed followed by a Student-Newman-Keuls post hoc test. ANOVA on rank followed by a Dunn’s post hoc test was performed when the data was not observing a normal distribution. Results are shown as mean ± SEM unless otherwise specified.

Results Low intensity pre-stimulus is required to elicit robust secretion We used TIRFM to investigate coupling of stimulation and secretion of LDCVs at the soma of mouse DRG neurons. LDCVs were specifically labelled by overexpression of NPY-Venus. 7 to 8 DIV old cells were maintained at 32 ± 2°C and stimulated either with 60 mM KCl or by using a field electrode while TIRFM movies were acquire at 10 Hz. To avoid Ca2+ channel inactivation, the solution containing 60 mM KCl was applied 3 times consecutively for 5 s every 20 s instead of one stimulation lasting 15 s. About 16% of the cells showed LDCV exocytosis (Table 1) and only the second stimulus induced strong release at a rate of 0.174 ± 0.004 s-1 (n=11, Fig. 3A). Some LDCV release could be measured prior to solution application. But because DRG neurons have been shown to be electrically silent when maintained in control medium (Ransom et al., 1977a), this LDCV release preceding the stimulus can only be explained by solution leak. To avoid this problem we chose to stimulate the neurons via field electrode and first tested which stimulation pattern was best to produce maximum LDCV exocytosis. The field electrode was set to 10 µA and 3 trains of stimulation, each lasting 30 s and separated by 20 s, were applied to the cells. We used two stimulation frequencies and a variety of pulse durations (Fig. 3B). The proportion of responding cells was variable (Table 1). Overall 33% of the cells responded to field electrode stimulation and only these cells were included in further analysis. We hypothesized that the non-responding cells were non-peptidergic neurons. In our culture condition they represented 46.9 ± 0.3% of all DRG neurons (Fig. 1D). If this were true then large neurons, which are peptidergic, should predominantly respond while small, which are nonpeptidergic, should not. However, somas of the recorded neurons had an area comprised between 140 and 450 µm2 for responding as well as for non-responding cells. This size range corresponds to medium sized DRG neurons that are 50% peptidergic and 50% non9

peptidergic (Stucky and Lewin, 1999). Thus it is likely that non-responding cells are essentially non-peptidergic neurons. No systematic correlation could be found between individual pulse duration or stimulation frequency and total secretion (Fig. 3D), nor between latency and secretion strength (Fig. 3E). Latency to secrete after stimulus onset varied between 19.6 ± 5.6 s for stimulation at 100 Hz with an individual pulse length of 4 ms (n=8) and 66.7 ± 15.4 s at a stimulation frequency of 100 Hz with an individual pulse length of 3 ms (n=14). No secretion was observed prior to stimulation but in most cases cells continued to secrete between stimulation trains (Fig. 3B). The cells, in which the secretion was best tuned to the stimulation periods, were those stimulated at 100 Hz with individual pulses lasting 3 ms. For this reason, we decided to use this stimulation paradigm for further study. We next stimulated cells for longer time periods to assess whether a pool of releasable vesicles could be exhausted in DRG neurons. As can be seen in Fig. 3C the cells secreted steadily at a linear rate of 0.037 ± 0.006 LDCVs∙s-1 throughout 2 min stimulation at 100 Hz (n=17). This is good evidence for the lack of a significant pool of ready to fuse LDCVs indicating that priming of LDCVs occurs at low rate in resting DRG neurons. It is well established that Ca2+ induces priming (Neher and Zucker, 1993, Voets, 2000, Neher and Sakaba, 2008) as well as docking (Pasche et al., 2012). Thus, we tested if an increase in intracellular Ca2+ concentration would reduce the latency between stimulus and secretion and if secretion could reach a plateau. For this we applied a pre-stimulus for 4 min at 5 Hz before stimulating the neurons at 100 Hz for 3 min. The pre-stimulus induced secretion at a very low rate of 0.005 ± 0.001 LDCVs∙s-1 while the stimulus at 100 Hz induced secretion that was best fitted with a mono-exponential growth with a time constant of 88 ± 25 s (Fig. 3C, n=17). The initial secretion rate was 0.056 ± 0.007 LDCVs∙s-1. Latency between start of stimulation and secretion, although not significant, was reduced from 37.8 ± 7.6 s without pre-stimulus to 28.3 ± 5.9 s with pre-stimulus (p=0.085; n = 17 for each condition). Applying a low frequency stimulus did not cause substantial LDCV secretion but enhanced to some degree secretion induced by high frequency stimulus. Full fusion exocytosis is the preferred mode of exocytosis in neurons stimulated via field electrode but not with 60 mM KCl. Previous reports showed that the main mode of brain derived neurotrophic factor (BDNF) loaded LDCV secretion in hippocampal neurons is kiss-and-run exocytosis, in which vesicles 10

fuse with the plasma membrane over a small fusion pore but in contrast to full fusion vesicles never fully collapse into the plasma membrane (Xia et al., 2009). To assess whether the mechanism of LDCV exocytosis in DRG and hippocampal neurons is similar, we used the pH sensitivity of Venus. With a pKa of 6 its fluorescence clearly increases when pH of LDCVS is neutralized via NH4+ application (Fig. 4A, see also (Nagai et al., 2002)). Hence, upon fusion pore opening Venus fluorescence should abruptly increase as LDCV lumen is neutralized. Because pH sensitivity of Venus is reduced in comparison to pHluorin (pKa=7.6) relatively small fluorescence intensity changes were observed. Nevertheless, they allowed us to differentiate between different fusion modes. We analyzed the fluorescence changes of individual LDCVs and their direct surroundings that reflects the release of NPY-Venus, for 3 s prior to and 3 s after fusion. We distinguished three clearly different progressions of the fluorescence intensity. In the first case fluorescence intensity of the LDCV was either high at the beginning of the experiment or it increased slowly over several seconds reaching a maximum, to then abruptly (less than 300 ms) drop returning to background level (Fig. 4B). We classified these full release types of events as full fusion. A second possibility (Fig. 4C) was that the fluorescence intensity of the LDCV was above background and abruptly increased in 0.16 ± 0.01 s before decreasing to background levels in 0.37 ± 0.05 s (Fig. 4C). In 86.8% of the cases the fusion events were accompanied by a small increase of the fluorescence in the surrounding of the LDCV (red trace) representing diffusion of NPY-Venus lasting at least 200 ms (Fig. 4C). These events were classified as kiss-and-run exocytosis. Finally, the fluorescence intensity of some vesicles that were present at the plasma membrane showed an abrupt and very transient increase. But, in contrast to kiss-and-run, their fluorescence intensity did not decrease to background level. Instead they reached similar fluorescence levels as before fusion (Fig. 4D). 92.3% of these events displayed an expansion of the fluorescence surrounding the LDCV, revealing partial release of NPY-Venus (Fig. 4D). Fast decreasing fluorescence intensity indicated the closure of the fusion pore and re-acidification of the LDCV thereby partially quenching the unreleased NPY-Venus. The fact that for these events the fluorescence did not decrease to background level indicated that the vesicle stayed at the plasma membrane for possibly a new round of exocytosis as shown in Fig. 4D. Thus they were classified as kiss-and-stay exocytosis. In more than 85% of the cases, the fluorescence of LDCVs changed slowly from barely visible to high intensity only few seconds prior to fusion. This slow increase in LDCVs’ fluorescence 11

intensity corresponds to the movement of vesicles traveling through the 300 nm thick evanescent wave from the cytoplasm to the plasma membrane for docking, ie tethering. To evaluate how long LDCVs remained close to the plasma membrane, we measured how much time elapsed between the appearance of the LDCV in the evanescent wave and its exocytosis. Independent of the stimulation method, the residency time of LDCVs undergoing full fusion was 20.7 ± 2.5 s and it was 25.4 ± 3.21 s for LDCVs exocytosed through kiss-andrun or kiss-and-stay fusion. However, the frequency distribution of residency time was strongly skewed and the median residency time was 2.8 s for full fusion and 4.6 s in case of kiss-and-run or kiss-and-stay fusion (Fig. 5E). This indicates that in resting DRG neurons LDCVs do not form a standing pool of docked vesicles. Another intriguing phenomenon was that in a large majority of full fusion events (about 73.4%) no transient increase of LDCV fluorescence due to NPY-Venus pH unquenching was observed and the fluorescence of the surrounding of the LDCV did not increase. The simplest explanation for this observation is that fusion pore expansion is so fast that NPY-Venus diffusion out of the LDCV competes with the unquenching of the dye and that the cloud of released NPY-Venus is diluted too quickly to be visualized. In contrast, in kiss-and-run or kiss-and-stay exocytosis the fusion pore opening visualized by the transient unquenching of NPY-Venus lasted 0.4 ± 0.02 s independent of the stimulus applied to the cell (Fig. 5B). This fusion pore probably remained narrow so that NPY-Venus slowly trickles out of the LDCVs thereby allowing the visualization of NPY-Venus pH unquenching and NPY-Venus cloud formation and dispersion. The stimulation method did not affect any of the fusion type properties (Fig. 5B, C and D). However, it strongly influenced the frequency at which they occurred. Field electrode stimulation of DRG neurons induced about 5 times more full fusion exocytosis than 60 mM KCl. Independent of the stimulation protocol the number of LDCVs secreted via kissand-run and kiss-and-stay exocytosis were 1.0 ± 0.2 and 0.29 ± 0.07 LDCVs, respectively (Fig. 5A). In other words, more than 65% of all events elicited by field electrode stimulation were full fusion exocytosis, whereas 60 mM KCl induced about equal amount of all fusion types. All three fusion types occurred evenly throughout the stimulation period, whether the cells were depolarized by 60 mM KCl or by field electrode stimulation (Fig. 5F). Thus the large difference in number of full fusion events was not due to the difference of the total stimulation time (60 mM KCl was applied intermittently for less than one minute and field electrode stimulation for more than 2 min). Furthermore, we tested how stimulation 12

protocols raised the intracellular Ca2+ concentration using Fura-2 measurements. The peak Fura-2 ratio was 0.16 ± 0.01 for all stimulus protocols (Fig. 6), suggesting that Ca2+ influx might not be responsible for increasing numbers of full fusion events elicited by field electrode stimulation as compared to 60 mM KCl application. However, we cannot rule out that the formation of Ca2+ microdomains may be different depending on the stimulation protocol. NPY increases LDCV exocytosis in DRG neurons It has been shown that the peptidergic content of LDCV regulates the excitability of DRG neurons (Ryu et al., 1988, Fjell et al., 1999, Leffler et al., 2002), but to our knowledge the effect of these neuromodulators on LDCV secretion has yet to be studied. We investigated the effect of NPY on the release of LDCVs. We used the following protocol: From an initial stimulation at 5 Hz, DRG neurons were stimulated twice at 100 Hz for 3 min with an interval of 6 min. During this interval, control cells remained untreated, whereas the others were incubated for 5 min with 50 nM NPY (Fig. 7A, right). LDCV secretion was measured for 1 min during the initial stimulation and for 3 min while cells were stimulated at 100 Hz. In control cells two consecutive trains of stimulation at 100 Hz appeared to deplete the pool of releasable LDCVs, because the number of secreted LDCVs detected during the second stimulus was reduced by about a factor of two in comparison to secretion during the first stimulation (Fig. 7A, left panel). When cells were treated with NPY no such depletion could be observed. Instead secretion was increased by about 30% (Fig. 7A). Thus, when comparing secretion occurring during the second round of stimulation, we found that NPY raised total secretion by a factor of 3.7. Although this was a large increase in secretion, statistical test failed to show a significant effect of NPY due to extremely high variability (n= 10 and 17 for control and NPY treated, respectively). NPY application did not affect the percentage of cells that secreted (26% and 28% of cells secreted prior and after NPY application, respectively) nor the average latency between start of stimulation and beginning of secretion (Fig. 7F). Reduction or increase of secretion in control or NPY-treated cells respectively, appears to be mainly due to changes in the number of full fusion events (Fig. 7B). Application of 50 nM NPY to the DRG neurons increased secretion of LDCVs but, this increase had an extremely high cell to cell variability as revealed by the large SEM (Fig. 7A). This might be due to variable expression levels of NPY receptors (YR) in DRG neurons. There are 5 13

known mammalian NPY receptors but only Y1R and Y2R are expressed in DRG neurons in rat and mouse (Hokfelt et al., 2007). To test whether the expression profile of YRs in our culture are responsible for the measurements variability, we performed immunocytochemistry to identify Y1R and Y2R expressing neurons. We found that 21.7 ± 9.5% DRG neurons expressed Y1R and 23.7 ± 7.5% expressed Y2R (± SD, ncell=141 and 173 respectively; Nculture=2 for both YR; Fig. 7G). The surface area of Y1R or Y2R positive neurons was 175.6 ± 13.8 µm2 and 183.4 ± 15.7 µm2, respectively (Fig. 7H). This surface area corresponded to the size of cells that were measured for LDCV secretion. This low percentage of Y1R or Y2R positive neurons explains well the variability of the effect on LDCV secretion induced by NPY application.

Discussion We examined the stimulation-secretion coupling of LDCVs in DRG neurons. For this we used a variety of stimuli and we found that 60 mM KCl or high frequency field electrode stimulation both induced secretion to a similar degree. Latency between secretion and stimulation was more than 20 s, which is comparable to what was measured for BDNF or atrial natriuretic peptide release from hippocampal neurons (Xia et al., 2009) and GABAergic hippocampal interneurons (Shinoda et al., 2011). Interestingly docking, which corresponds to the dwell time of the LDCVs close to the membrane prior to fusion, was very short (less than 10 s for more than 60% of the LDCVs) independent of their fusion mode, indicating that in resting DRG neurons LDCVs neither form a docked pool nor a readily releasable pool. This corresponds to a finding by Huang and Neher (1996) in which they showed that an application of ionomycin to DRG neurons induces a secretory response delayed by several seconds after [Ca2+]i was raised. However, they also showed that depolarizing DRG neurons for 100 – 200 ms induces a fast stepwise increase in membrane capacitance reflecting fast secretion and the existence of a readily releasable pool of vesicles. Additionally, we showed that low frequency pre-stimulus, which mildly increases [Ca2+]i, did not increase the dwell time of LDCVs close to the membrane prior to fusion. This suggests that, unlike in chromaffin cells, docking is not significantly facilitated through high [Ca2+]i in DRG neurons (Pasche et al., 2012). Conversely, the same low frequency stimulus was able to reduce to some extend the latency between secretion and stimulation. Thus a mild increase in [Ca2+]i might promote LDCV priming but not docking in DRG neurons. In vivo, DRG neurons show spontaneous 14

activity at 0.5 to 5 Hz while stimulation induces bursting or tonic activity to at least 50 Hz (Wall and Devor, 1983). Based on our data, we can conclude that in a healthy physiological situation very few LDCVs are secreted. However, the basal stimulation probably induces effective priming conditions so that a large pool of LDCVs is ready to be released when the neurons receive strong sensory inputs or in pathological situations. Indeed, after nerve injury DRG neurons fire action potentials of ectopic origin at high frequency (Michaelis et al., 2000), which might be sufficient to induce LDCV release. Furthermore, we analyzed the mode of secretion. We distinguished between three types of fusion, full fusion, kiss-and-run and kiss-and-stay exocytosis (Harata et al., 2006) according to change of the LDCV fluorescence intensity during exocytosis. NPY-Venus is relatively small and diffuses rapidly in the extracellular space upon secretion (Taraska et al., 2003, Perrais et al., 2004) unlike other peptides bound to a fluorescence protein, such as tissue plasminogen activator, semaphoring 3A or BDNF (Brigadski et al., 2005, de Wit et al., 2009). Hence, full fusion of LDCVs is accompanied by full release of NPY-Venus that results in very fast decay down to background level of their fluorescence intensity (Perrais et al., 2004, de Wit et al., 2009). Reuptake of LDCV content as described by Perrais et al. (2004) is very unlikely if the LDCV label is NPY-Venus (Taraska et al., 2003). Partial release of NPY-Venus that is marked by a cloud of dye is certainly due to transient opening of a fusion pore without full collapse of the vesicle in the plasma membrane (Tsuboi and Rutter, 2003). In conclusion, the best explanation for full and partial release of NPY-Venus is that they are likely due to two different mechanisms namely full fusion and kiss-and-run or kiss-and-stay exocytosis. Both kiss-and-run and kiss-and-stay exocytosis are mechanistically very similar in that they are transient. This is supported by the fact that all characteristics of fluorescence changes during exocytosis (peak width, rise and decay time, Fig. 5) are identical for kiss-and-run and kiss-and-stay. The only difference is that in one case the vesicle leaves the plasma membrane immediately after closure of the fusion pore, while in the other case it remain close to it. Nevertheless, kiss-and-stay exocytosis indicates that DRG neurons are able to fine tune the amount of peptide release by regulating the amount of release from single vesicles as it has been shown for BDNF release in hippocampal neurons (Lessmann and Brigadski, 2009). If we pool both transient fusion types together, we find that full fusion is the preferred mode of LDCV exocytosis in DRG neurons stimulated by field electrode (Fig. 5A). On the contrary, if neurons were depolarized via 60 mM KCl application then transient fusion 15

(about 1.6 event per neuron) was clearly favored over full fusion (about 0.5 event per neuron). The low rate of full fusion events induced by 60 mM KCl is in full agreement with the fusion type observed in hippocampal neurons stimulated by application of a solution containing high [K+] (Xia et al., 2009). However, when hippocampal neurons were stimulated at 10 Hz via field-electrode LDCVs were released via kiss-and-run at the axon but mainly via full fusion at dendrites (Matsuda et al., 2009). This discrepancy in fusion modes between field electrode stimulation and depolarizing KCl was not found in cortical neurons (de Wit et al., 2009). Taken together, exocytosis at the soma of DRG neurons resembles that of hippocampal dendrites. Because field electrode stimulation emulates physiological stimuli to a better degree, we hypothesize that LDCV exocytosis from neurons in vivo occurs primarily through full fusion. Treating DRG neurons with NPY increased secretion of LDCVs by more than a factor of 3. However, the degree to which LDCV secretion was increased after NPY application was extremely variable. We found that less than a quarter of neurons were positive for either NPY receptor subtype, which compares well with previous literature. In rat about 25% to 40% of DRG neurons express Y1 receptors and 10% to 40% express Y2 receptors (Zhang et al., 1995, Brumovsky et al., 2005, Landry et al., 2005, Hokfelt et al., 2007). In mouse only a small percentage of DRG neurons are Y1- or Y2-positive (6% and 15%, respectively, Shi et al. (1998); 20% were found to be Y1-positive by Wiley et al. (2009)). After axotomy the percentage of Y1-positive neurons does not change but Y2 positive neurons increase (Shi et al., 1998). Our data does not agree with data from Hiruma et al. (2002), showing that up to 85% of the DRG neurons in culture expressed Y1 receptors. Hence, our data support the idea that a highly variable response of DRG neurons to NPY application can be explained by a low percentage of neurons expressing NPY receptors. Finally, almost all Y1R-positive neurons have been shown to be peptidergic neurons, while only 40% of the Y2R-positive neurons are peptidergic (Hokfelt et al., 2007). We speculate that DRG neurons that secrete LDCVs upon electrical stimulation are peptidergic neurons. If Y1R activation would promote LDCV exocytosis then it should have been promoted in a majority of responding DRG neurons. Thus we hypothesize that NPY application to DRG neurons enhances stimulated LDCV secretion through activation of Y2R. Under normal physiological conditions only few DRG neurons express NPY (Schoenen et al., 1989) and our study demonstrates that LDCV exocytosis is low as well. However, after nerve 16

injury, which is mimicked by in vitro conditions, the number of DRG neurons that express NPY increases dramatically (Schoenen et al., 1989, Boateng et al., 2015, Magnussen et al., 2015). At the same time the expression of Y2R, which have been shown to promote DRG neurons excitability (Abdulla and Smith, 1999), is raised as well (Zhang et al., 1997). Therefore, our results suggest that NPY induces a positive feedback mechanism in vivo after nerve lesion, reinforcing its release. Since NPY exerts an anti-nociceptive effect (Solway et al., 2011), this autocrine and possibly paracrine signaling of NPY that has been proposed by Hokfelt et al. (2007) can effectively reduce chronic pain.

Conflict of Interest: The authors declare that they have no competing interests. Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB894 and GK1326 U.B. and B.N.). A. B. and A.S. are members of the DFG-sponsored graduate program GK1326. We thank Jens Rettig and David Stevens for helpful comments on the manuscript, Margarete Klose for expert technical support and Detlef Hof for his support in programming. Author contribution: U.B. conceived the experiments which were co-designed by A.B.. A.B. and A.S. executed and interpreted the findings. Y.S. provided the Lenti Virus. U.B. and B.N. wrote the article. All authors revised the article.

References Abdulla FA, Smith PA (1999) Nerve injury increases an excitatory action of neuropeptide Y and Y2agonists on dorsal root ganglion neurons. Neuroscience 89:43-60. Becherer U, Pasche M, Nofal S, Hof D, Matti U, Rettig J (2007) Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells. PloS one 2:e505. Becherer U, Rettig J (2006) Vesicle pools, docking, priming, and release. Cell and tissue research 326:393-407. Boateng EK, Novejarque A, Pheby T, Rice AS, Huang W (2015) Heterogeneous responses of dorsal root ganglion neurons in neuropathies induced by peripheral nerve trauma and the antiretroviral drug stavudine. Eur J Pain 19:236-245. Brigadski T, Hartmann M, Lessmann V (2005) Differential vesicular targeting and time course of synaptic secretion of the mammalian neurotrophins. J Neurosci 25:7601-7614.

17

Brumovsky P, Stanic D, Shuster S, Herzog H, Villar M, Hokfelt T (2005) Neuropeptide Y2 receptor protein is present in peptidergic and nonpeptidergic primary sensory neurons of the mouse. The Journal of comparative neurology 489:328-348. de Wit J, Toonen RF, Verhage M (2009) Matrix-dependent local retention of secretory vesicle cargo in cortical neurons. J Neurosci 29:23-37. Farina M, van de Bospoort R, He E, Persoon CM, van Weering JR, Broeke JH, Verhage M, Toonen RF (2015) CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons. Elife 4. Fjell J, Cummins TR, Dib-Hajj SD, Fried K, Black JA, Waxman SG (1999) Differential role of GDNF and NGF in the maintenance of two TTX-resistant sodium channels in adult DRG neurons. Brain research Molecular brain research 67:267-282. Fulop T, Radabaugh S, Smith C (2005) Activity-dependent differential transmitter release in mouse adrenal chromaffin cells. J Neurosci 25:7324-7332. Guzman RE, Schwarz YN, Rettig J, Bruns D (2010) SNARE force synchronizes synaptic vesicle fusion and controls the kinetics of quantal synaptic transmission. J Neurosci 30:10272-10281. Harata NC, Aravanis AM, Tsien RW (2006) Kiss-and-run and full-collapse fusion as modes of exoendocytosis in neurosecretion. Journal of neurochemistry 97:1546-1570. Hiruma H, Saito A, Kusakabe T, Takenaka T, Kawakami T (2002) Neuropeptide Y inhibits axonal transport of particles in neurites of cultured adult mouse dorsal root ganglion cells. The Journal of physiology 543:85-97. Hokfelt T, Brumovsky P, Shi T, Pedrazzini T, Villar M (2007) NPY and pain as seen from the histochemical side. Peptides 28:365-372. Hokfelt T, Johansson O, Ljungdahl A, Lundberg JM, Schultzberg M (1980) Peptidergic neurones. Nature 284:515-521. Huang LY, Neher E (1996) Ca(2+)-dependent exocytosis in the somata of dorsal root ganglion neurons. Neuron 17:135-145. Kubo A, Katanosaka K, Mizumura K (2012) Extracellular matrix proteoglycan plays a pivotal role in sensitization by low pH of mechanosensitive currents in nociceptive sensory neurones. The Journal of physiology 590:2995-3007. Landry M, Liu HX, Shi TJ, Brumovsky P, Nagy F, Hokfelt T (2005) Galaninergic mechanisms at the spinal level: focus on histochemical phenotyping. Neuropeptides 39:223-231. Leffler A, Cummins TR, Dib-Hajj SD, Hormuzdiar WN, Black JA, Waxman SG (2002) GDNF and NGF reverse changes in repriming of TTX-sensitive Na(+) currents following axotomy of dorsal root ganglion neurons. Journal of neurophysiology 88:650-658. Lessmann V, Brigadski T (2009) Mechanisms, locations, and kinetics of synaptic BDNF secretion: an update. Neuroscience research 65:11-22. Magnussen C, Hung SP, Ribeiro-da-Silva A (2015) Novel expression pattern of neuropeptide Y immunoreactivity in the peripheral nervous system in a rat model of neuropathic pain. Mol Pain 11:31. Matsuda N, Lu H, Fukata Y, Noritake J, Gao H, Mukherjee S, Nemoto T, Fukata M, Poo MM (2009) Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite. J Neurosci 29:14185-14198. Michaelis M, Liu X, Janig W (2000) Axotomized and intact muscle afferents but no skin afferents develop ongoing discharges of dorsal root ganglion origin after peripheral nerve lesion. J Neurosci 20:2742-2748. Murakami M, Fleischmann B, De Felipe C, Freichel M, Trost C, Ludwig A, Wissenbach U, Schwegler H, Hofmann F, Hescheler J, Flockerzi V, Cavalie A (2002) Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels. The Journal of biological chemistry 277:40342-40351. Murase K, Randic M (1984) Actions of substance P on rat spinal dorsal horn neurones. The Journal of physiology 346:203-217.

18

Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87-90. Neher E, Sakaba T (2008) Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron 59:861-872. Neher E, Zucker RS (1993) Multiple calcium-dependent processes related to secretion in bovine chromaffin cells. Neuron 10:21-30. Pasche M, Matti U, Hof D, Rettig J, Becherer U (2012) Docking of LDCVs Is Modulated by Lower Intracellular [Ca2+] than Priming. PloS one 7:e36416. Perrais D, Kleppe IC, Taraska JW, Almers W (2004) Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells. The Journal of physiology 560:413-428. Pezet S, McMahon SB (2006) Neurotrophins: mediators and modulators of pain. Annual review of neuroscience 29:507-538. Quintana A, Kummerow C, Junker C, Becherer U, Hoth M (2009) Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals. Cell calcium 45:109-122. Ramamoorthy P, Wang Q, Whim MD (2011) Cell type-dependent trafficking of neuropeptide Ycontaining dense core granules in CNS neurons. J Neurosci 31:14783-14788. Randic M, Hecimovic H, Ryu PD (1990) Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones. Neuroscience letters 117:74-80. Ransom BR, Christian CN, Bullock PN, Nelson PG (1977a) Mouse spinal cord in cell culture. II. Synaptic activity and circuit behavior. Journal of neurophysiology 40:1151-1162. Ransom BR, Neale E, Henkart M, Bullock PN, Nelson PG (1977b) Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties. Journal of neurophysiology 40:1132-1150. Ryu PD, Murase K, Gerber G, Randic M (1988) Actions of calcitonin gene-related peptide on rat sensory ganglion neurones. Physiologia Bohemoslovaca 37:259-265. Schoenen J, Delree P, Leprince P, Moonen G (1989) Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: an immunocytochemical study. Journal of neuroscience research 22:473-487. Shi TJ, Zhang X, Berge OG, Erickson JC, Palmiter RD, Hokfelt T (1998) Effect of peripheral axotomy on dorsal root ganglion neuron phenotype and autonomy behaviour in neuropeptide Y-deficient mice. Regulatory peptides 75-76:161-173. Shinoda Y, Sadakata T, Nakao K, Katoh-Semba R, Kinameri E, Furuya A, Yanagawa Y, Hirase H, Furuichi T (2011) Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network. Proceedings of the National Academy of Sciences of the United States of America 108:373-378. Solway B, Bose SC, Corder G, Donahue RR, Taylor BK (2011) Tonic inhibition of chronic pain by neuropeptide Y. Proceedings of the National Academy of Sciences of the United States of America 108:7224-7229. Sorensen JB (2004) Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles. Pflugers Arch 448:347-362. Stucky CL, Lewin GR (1999) Isolectin B(4)-positive and -negative nociceptors are functionally distinct. J Neurosci 19:6497-6505. Taraska JW, Perrais D, Ohara-Imaizumi M, Nagamatsu S, Almers W (2003) Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proceedings of the National Academy of Sciences of the United States of America 100:2070-2075. Tsuboi T, Rutter GA (2003) Multiple forms of "kiss-and-run" exocytosis revealed by evanescent wave microscopy. Current Biology 13:563-567.

19

van de Bospoort R, Farina M, Schmitz SK, de Jong A, de Wit H, Verhage M, Toonen RF (2012) Munc13 controls the location and efficiency of dense-core vesicle release in neurons. The Journal of cell biology 199:883-891. Voets T (2000) Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices. Neuron 28:537-545. Wall PD, Devor M (1983) Sensory afferent impulses originate from dorsal root ganglia as well as from the periphery in normal and nerve injured rats. Pain 17:321-339. Wiley RG, Lemons LL, Kline RHt (2009) Neuropeptide Y receptor-expressing dorsal horn neurons: role in nocifensive reflex responses to heat and formalin. Neuroscience 161:139-147. Xia X, Lessmann V, Martin TF (2009) Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events. Journal of cell science 122:7582. Zhang C, Zhou Z (2002) Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons. Nature neuroscience 5:425-430. Zhang X, Aman K, Hokfelt T (1995) Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy. The Journal of comparative neurology 352:481-500. Zhang X, Shi T, Holmberg K, Landry M, Huang W, Xiao H, Ju G, Hokfelt T (1997) Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia. Proceedings of the National Academy of Sciences of the United States of America 94:729-734. Zheng H, Fan J, Xiong W, Zhang C, Wang XB, Liu T, Liu HJ, Sun L, Wang YS, Zheng LH, Wang BR, Zhang CX, Zhou Z (2009) Action potential modulates Ca2+-dependent and Ca2+-independent secretion in a sensory neuron. Biophysical journal 96:2449-2456.

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Figure Legends Figure 1: DRG neurons can be distinguished by size and shape from non-neural cells. A, Picture from a DRG neuron expressing NPY-Venus. In the bright field image (Ai) a thick network of neurites can be distinguished as well as a non-neural cell (arrow). The shadow of the field electrode is visible in the upper part of the image. Epifluorescence (Aii) and TIRFM (Aiii) images of the cells show clearly the NPY-Venus dotted staining pattern. Analyze of LDCVs secretion was performed at the cell soma. B, Epifluorescence image of an astrocyte transfected with NPY-Venus. The cell shape and labeling pattern are clearly different to DRG neurons. When such a cell was visible then the entire field of view was rejected. C, Transfected Schwann cell are easily distinguished from DRG neurons. The density of Schwann cells in our culture was low an only occasionally they became infected with the Lenti virus. Their shape as can be seen in the bright field image (Ci) was very different to neurons, and their NPY-Venus staining pattern visible in the epifluorescent image (Cii) was clearly more diffuse than in neurons. The inset shows the cell body at unsaturating brightness settings. D, Image of DRG neurons acquired in brightfield illumination (left) and epifluorescent at 561 nm (right) to visualize the IB4 labeling. Neurons with bright red cell membrane were IB4-positive non-peptidergic neurons, while unstained neurons were peptidergic neurons. ii, IB 4 positive and negativ neurons expressed as percent of total number of neuron (n cells=147, Ncultures=2).

Figure 2: Classification criteria of different LDCV fusion modes A, Depicted are the change of fluorescent intensity over time of 3 imaginary vesicles undergoing exocytosis either through full fusion (green), kiss-and-run (red) or kiss-and-stay (blue) to illustrate the 3 selection criteria used to differentiate between fusion modes.
Identification of exocytosis. At the time of exocytosis, the LDCV fluorescent intensity should be at its own maximum and at least 4 times higher than background, before abruptly (in 200 ms) decreasing. If this criterium was not met then the vesicle was not fusing with the plasma membrane and the spot was discarded.  Distinction between full fusion and the other fusion modes. First the vesicles fluorescence intensity just after fusion was compared to the local background. It should be reached in less than 300 ms for full fusion. Second, to confirm this classification we analyzed the fluorescence intensity rise time prior to fusion. If it was slow or not existent (in the measured time window of 3 s) then the event was classified as 21

full fusion. If instead the increase in fluorescent intensity was larger than 3 standard deviation (SD) of its own fluorescence fluctuation in less than 325 ms (see panel B) then the event was categorized as kiss-and-run or kiss-and-stay.  Distinction between kiss-and-run and kiss-and-stay. The average fluorescence intensity over 1 s, 3 s before and after fusion of the vesicle was measured and compared. If the vesicle fluorescence intensity after fusion was reduced by more than 4 SDs of its intensity fluctuation in comparison to the fluorescence before fusion, then the fusion event was classified as kiss-and-run. Else the event was a kiss-and-stay event. B, Histogram of the fluorescent rise time of all LDCVs shown in Fig. 5C. It was best fit by a sum of two lognormal. pH dequenching of Venus upon fusion pore opening induced a rise time of LDCV fluorescence intensity that was comprised in the first lognormal. In contrast the increase in fluorescence intensity that goes along with the movement of LDCVs to the plasma membrane during docking had a rise time included in the second lognormal. The intersection point between both fit was 325 ms, which was used as criterion‚ in panel A.

Figure 3: LDCV fusion in DRG neurons can be evoked both with high [K+] and electrically via field electrode stimulation. A-C, Average cumulative LDCV release induced by different types of stimulation in responding DRG neurons. A, LDCV exocytosis triggered by application of a solution containing 60 mM K+ (Ncells=11). B, Repetitive short term stimulations with a field electrode (10 µA amplitude) using several pulse durations and frequencies (3 ms at 50 Hz Ncells=10; 9 ms at 100 Hz Ncells=9; 2 ms at 100 Hz Ncells= 2; 3 ms at 100 Hz Ncells=14; 4 ms at 100 Hz Ncells=8). C, Long term stimulation with a field electrode (10 µA amplitude, 3 ms pulse duration at 100 Hz) with and without 4 min prestimulus (10 µA amplitude, 3 ms pulse duration at 5 Hz) (Ncells=17 for both conditions). D, Average total secretion of the cells shown in A-C. Bars labelled all/50 and all/100 correspond to the pooled average latencies of all field electrode stimulations at 50 Hz and 100 Hz, respectively. E, Average latency of the first LDCV exocytosed after the onset of stimulation of the cells shown in A-C.

22

Figure 4: Fusion events of LDCVs in DRG neurons can be classified in 3 different types: full fusion (B), kiss-and-run (C) and kiss-and-stay (D). A, The fluorescent protein Venus is quenched by low pH. 5 s after recording started DRG neurons expressing NPY-Venus were superfused with a ringer solution containing 40 mM NH4+ to deprotonate LDCV lumen. Venus responded to the pH change by an increase in fluorescence intensity as shown on the right hand graph of normalized fluorescent values over time (Ncells=4). The extent of the fluorescence change can be appreciated on the pictures of an exemplary cell taken at the time points (a, b, c) indicated on the graph. The fluorescence intensity scale is displayed on the left. B, Example of a typical full fusion event. The upper row corresponds to sequential images of the LDCV prior to (a) and during (b-c) fusion. Corresponding fluorescence intensity traces of the areas of the secreted vesicle (yellow line) and around it (red line, see inset) is shown below. Arrows indicate the time points in the fluorescence trace at which individual images and the first and last pictures of the sequence were acquired. C, Exemplary sequential images and corresponding fluorescence trace of a fusion event, defined as kiss-and-run. In the first image (a) the LDCV is faintly visible at the plasma membrane 3 s prior to exocytosis. The sequence (b-c) shows the fusion event. The last image (d) corresponds to the empty membrane area after the vesicle moved back in the cytoplasm. D, Exemplary image sequence of fusion events defined as kiss-and-stay. The first 2 images (a, b) show the LDCVs as it approaches the plasma membrane for tethering. (c-d) and (e-f) are two sequences depicting the same LDCV fusing twice. The image acquisition frequency was 10 Hz. Scale bar in A is 5 µm and 2 µm in B, C and D.

Figure 5: Full fusion is the preferred mode of exocytosis of LDCVs in DRG neurons stimulated with field electrode but not with high [K+]. A, Average number of fusion events in the three modes of exocytosis (50 Hz: nLDCV =75; 100 Hz: nLDCV =74; 3 ms/100 Hz/2 min: nLDCV =70; 3 ms/100 Hz/3 min: nLDCV =100; 60 mM K+: nLDCV =20). Changes in the fluorescence intensity of each LDCV during exocytosis was analyzed to characterize the fusion type. A time window of 3 s before and after fusion was used for this analysis as indicated in the insets of panels B to D. B, Average peak width at half peak maximum of for kiss-and-run and kiss-and-stay fusion events (see inset), corresponding to fusion pore opening time. C, Average rise time of fluorescence intensity for kiss-and-run 23

and kiss-and-stay fusion type (for more details see inset and materials and methods). D, Average decay time of fluorescence intensity, which essentially equates to LDCV discharge after fusion pore opening (for more details see inset and materials and methods). E, Frequency distribution of the time LDCVs spend in the TIRF field prior to secretion. Solid or stippled lines correspond to LDCVs secreted via full fusion, or kiss-and-run and kiss-and-stay, respectively. Inset shows the frequency distribution of LDCVs staying close to the membrane for less than 15 s. F, Average cumulative number of secreted LDCVs over time subdivided into their fusion modes. All cells stimulated with field electrode were pooled and compared with cells stimulated with high [K+] application (field electrode stimulation: nLDCV for full fusion, kiss-and-run and kiss-and-stay was 219, 82 and 19 respectively; high [K+] stimulation: nLDCV for full fusion, kiss-and-run and kiss-and-stay was 6, 8 and 7). Inset shows the first 30 s of the same traces normalized to the total number of secreted LDCVs. *** p ≤ 0.001; * p ≤ 0.05.

Figure 6: 60 mM KCl and field electrode stimulation induced similar increase of [Ca2+]i Fura-2 measurements comparing Ca2+-responses of DRG neurons induced by different types of stimulation. Stimulation protocol is shown on top. Solid black line indicates time of high [K+] or strong (50 or 100 Hz) field electrode stimulation. Stippled line corresponds to 5 Hz prestimulation period for recordings shown in navy blue.

Figure 7: NPY sensitizes DRG neurons to lower frequency stimulation. A, Cumulative average LDCV secretion in two consecutive measurements in which cells were stimulated with a prestimulus at 5 Hz before being stimulated at 100 Hz for 3 min (individual pulse amplitude was 10 µA lasting 3 ms). 50 nM NPY was applied to cells for 5 min before the second round of stimulation (Ncells =17), while control cells were recorded without NPY application (Ncells =10). The protocol is depicted on top of corresponding traces. B-E, Show in depth analysis of fusion events (for details see Fig. 5; control nLDCV =62; movie 2 –NPY nLDCV =9; movie 2 + NPY nLDCV =58). B, Average number of fusion events. C, Average peak width. D, Average rise time. E, Average decay time. F, Average latency of the first fusion event. Data obtained during the first round of stimulation (movie 1) were pooled together and termed “control” and are compared to results obtained during the second round of stimulation (movie 2) in which cells were treated with NPY (+NPY) or not (-NPY). G, 24

Evaluation

of

NPY

receptors

expression

in

DRG

neurons

in

culture

with

immunocytochemistry of Y1R and Y2R. Representative pictures of neurons stained with antiY1R (top) and anti-Y2R (bottom) antibodies. Counting stained cells resulted in 21.7 ± 9.5% expressing Y1R and 23.7 ± 7.5% Y2R (± SD, ncells=141 and 173 respectively; Ncultures=2 for both YR). H, Surface area of the middle cross section of DRG neurons expressing either Y1R or Y2R. Each dot represents one neuron. Average size of neurons expressing Y1R or Y2R were 175.6 ± 13.8 µm2 and 183.4 ± 15.7 µm2, respectively (ncells= 31 and 40, respectively).

Table 1: Proportion of responding DRG neurons as a function of the applied stimulus 60 mM KCl

3 ms 50 Hz

9 ms 50 Hz

2 ms 100 Hz

3 ms 100 Hz

4 ms 100 Hz

3ms 100Hz 2 min

3 ms 100 Hz 3 min +prestim.

measured cells

66

31

23

14

37

19

43

39

responding cells (1)

11

10

09

02

14

08

17

17

39.5

43.6

Number of:

% of responding cells 16.6 32.3 39.1 14.3 37.8 42.1 (1) Only responding cells were represented in all figures of the present work

Highlights •

We studied large dense core vesicle exocytosis in DRG neurons with TIRF-microscopy.

•

Full or partial release of LDCV content is stimulus-dependent.

•

LDCV exocytosis is regulated via a positive feedback mechanism of NPY

•

This feedback might occur in vivo upon nerve lesion and thus reduce chronic pain.

25

Ai

ii

field electrode

iii

10 µm

10 µm

B

Ci

Di

10 µm

ii

10 μm

iB4-Alexa561

Percentage of DRG subytpes

10 µm

ii

100 80 60 40 20 0 Peptidergic Non-peptidergic

‚ rise time

Fluorescence intensity

> 350ms >

K&S K&R full fusion local bkg

B



<200 ms !<4×bkg

35

25

ƒ fluo.drop < 4 SD

SD

> 4SD

SD SD

‚ ≈ 0

325 ms

30

1

2

3 Time [s]

Occurrence

A

20 15 10 5 0

4

5

6

0

1 2 3 4 Fluorescence intensity rise time [s]

5

B

A

C

Cumulative # of fusion events

7

100 Hz 5 Hz 0 Hz

high Hz 0 Hz

6

+

3 ms / 50 Hz 9 ms / 50 Hz 2 ms / 100 Hz 3 ms / 100 Hz 4 ms / 100 Hz

60 mM K

5 4

3 ms / 100 Hz / 2 min 3 ms / 100 Hz 3 min + pre

3 2 1 0 0

0

20

40

60

0

80 100 120 140 160 180 Time [s]

20

E

10

40

60

80 100 120 140 160 180 200 220 240 Time [s]

120 Latency of 1st event [s]

Total fusion events [# LDCVs/cell]

D

20 40 60 Time [s]

8 6 4 2 0

0 3/5

+ in 0 0 0 0 0 0 re K h 9/5 all/5 2/10 3/10 4/10 ll/10 /2m in+p a hig 00 m 3/1 00/3 3/1

100 80 60 40 20 0

0 3/5

+ in 0 0 0 0 0 0 re K h 9/5 all/5 2/10 3/10 4/10 ll/10 /2m in+p a hig 00 m 3/1 00/3 3/1

4000 3000

Fluorescence intensity [A.U.]

c

b

c

0.6 0.4 0.2

a

0

5 µm

a

5

10 15 Time (s)

20

b

25

c

700

b

600 500

a

400

c

300 200 100 0

C

0

1

a

2

3 Time [s]

4

5

b

c

6

d

600 500 400 b

a

300

c

d

200 100 0 0

D

1

2

b

a

2nd

Fluorescence Intensity [A.U.]

0.8

0.0

1000

B

Fluorescence intensity [A.U.]

b

2000

NH4+

1.0

a

5000

(F-F0)/(Fmax-F0)

Fluorescence intensity [AU]

A

1st

3 Time [s]

4

5

c

6

d

e

f

1st

350

b

300

c

a

250

d

2nd e f

200 150 100 50 0

0

1

2

3 Time [s]

4

5

6

B *

# of fusion events

5.0

*

*** *

50 Hz 100 Hz / 3 × 30 s 3 ms / 100 Hz / 2 min 3 ms / 100 Hz / 3 min + pre + 60 mM K

4.0

1.0 0.8

3.0 2.0

0.6

Δt @ 50%

400 200 0

0

4 2 Time [s]

6

0.2

full fusion

kiss-&-run

D

0.8 0.6

600 10% 400 Δt 200

10%

0 0

4 2 Time [s]

1.0 0.8

6

0.4

Decay time [s]

Fluo intensity [AU]

1.0

0.0

kiss-&-stay

0.2

0.6

Fluo intensity [AU]

0.0

Rise time [s]

600

0.4

1.0

C

Fluo intensity [AU]

6.0

Peak width [s]

A

600

kiss-&-run

kiss-&-stay

kiss-&-run

kiss-&-stay

10%

400

Δt

200

10%

0 0

4 2 Time [s]

6

0.4 0.2

0.0

0.0

kiss-&-stay

kiss-&-run

full fusion kiss-&-run; kiss-&-stay

120

60 Occurence

Occurence

100 80 60 40

40 20 0 1 3 5 7 9 11 13 15 Time [s]

20

0

3.0 2.5 2.0

Norm. # of LDCV

F 140

Cumulative # of fusion events

E

full fusion

1.5

1

0

0

10 20 Time [s]

1.0

Field- 60 mM elect. KCl

full fusion kiss-&-run kiss-&-stay

0.5 0.0

0

50 100 Residency time [s]

150

0

20

40

60

80 100 120 140 160 Time [s]

protocol

Fura-2 ratio [350/380 nm]

1.25 1.20 1.15 1.10 1.05 1.00 0.95

3 ms / 50 Hz 3 ms / 100 Hz 3 ms / 100 Hz + pre high K+

0.90 0.85

0

50

100

150 Time [s]

200

250

300

A movie 5 min. incubation 100 Hz 5 Hz

Cumulative # of fusion events

7 6 5

movie 1 control movie 1 before NPY

4

movie 2 –NPY movie 2 +NPY

3 2 1 0 40

80

B

120 160 Time [s]

200

3 2

80

120 160 Time [s]

200

240

0.6

0.4

0.2

1 full fusion

kiss-&-run

D

0.0

kiss-&-stay

kiss-&-run

kiss-&-stay

E 0.6

Decay time [s]

0.6 0.4 0.2 0.0

kiss-&-stay

kiss-&-run

G

F

Anti Y1R

80 60

40 µm

40 20 0

control movie2 movie2 –NPY +NPY

40 µm

**

0.4 0.2 0.0

Bright field

100

Anti Y2R

Rise time [s]

40

C control movie 2 –NPY movie 2 +NPY

4

0

Latency of 1st event [s]

0

full fusion

kiss-&-run

Confocal 561 nm

kiss-&-stay

H Neuron surface area [µm2]

# of fusion events

5

240

Peak width [s]

0

500 400 300 200 100 0

Y1R Y2R