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Complexins are SNARE-complex binding proteins essential for the Ca2+-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a co-factor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca2+-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin-adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.
Four synaptotagmins (Syt1, Syt2, Syt7, and Syt9) function as Ca2+-sensors for synaptic and neuroendocrine vesicle exocytosis (Fernandez-Chacon et al., 2001; Sorensen et al., 2003; Xu et al., 2007; Gustavsson et al., 2009; Schonn et al., 2009), and require as co-factors complexins, which are small proteins that bind to SNARE complexes (McMahon et al., 1995 Reim et al., 2001). Complexins perform both activating and clamping functions in exocytosis, and their role is conserved from worms to mammals (e.g., see Reim et al., 2001; Tang et al., 2006; Huntwork and Littleton, 2007; Xue et al., 2007, 2009, and 2010; Cai et al., 2008; Maximov et al., 2009; Strenzke et al., 2009; Yang et al., 2010; Cho et al., 2010; Hobson et al., 2011; Martin et al., 2011; Giraudo et al., 2006, 2008, and 2009; Schaub et al., 2006; Yoon et al., 2008; Malsam et al., 2009; Seiler et al., 2009; Li et al., 2011; Krishnakumar et al., 2011). Structurally, complexins contain unstructured N- and C-terminal sequences that flank an accessory and a central α-helix (Chen et al., 2002; Bracher et al., 2002). The N-terminal complexin region is essential for activating synaptic vesicle exocytosis (Xue et al., 2007; Maximov et al., 2009) and for synaptic vesicle priming (Yang et al., 2010), the accessory α-helix of complexins is required for clamping synaptic vesicle exocytosis (Maximov et al., 2009), the central α-helix binds to SNARE complexes and is necessary for all complexin functions (Tang et al., 2006; Maximov et al., 2009; Martin et al., 2011), and the C-terminal sequence is required for the clamping and priming, but not for the synaptotagmin-activating functions of complexin (Kaeser-Woo et al., 2012). To date, all studies of complexin function focused on Ca2+-triggered exocytosis of synaptic vesicles and of large dense-core vesicles (LDCVs) that are controlled by four closely related synaptotagmins (Syt1, Syt2, Syt7, and Syt9; Xu et al., 2008; Gustafsson et al., 2008; Schonn et al., 2008). However, other types of Ca2+-regulated exocytosis may co-exist in neurons in addition to synaptic vesicle and LDCV exocytosis, and other isoforms of synaptotagmin are co-expressed with Syt1, Syt2, Syt7, and/or Syt9 in neurons. Indeed, we recently characterized in olfactory bulb neurons a previously unknown pathway of Ca2+-dependent exocytosis that is selectively controlled by Syt10 instead of Syt1, Syt2, Syt7, and/or Syt9, and that mediates IGF-1 secretion (Cao et al., 2011). Although much is known about complexin function in synaptic and LDCV exocytosis, it is unclear whether complexin also plays a role in other types of exocytosis that are controlled by more distantly related synaptotagmins.
Here, we examined a possible role for complexins in Ca2+-triggered IGF-1 exocytosis in olfactory bulb neurons that is regulated by Syt10 (Cao et al., 2011). We show that the Syt10-dependent secretory pathway of IGF-1 is distinct from the LDCV pathway of neuropeptide secretion, and that complexins are essential for both the Syt1-dependent pathway of neurotransmitter release and the Syt10-dependent pathway of IGF-1 secretion. Thus, complexins represent a conserved component of distinct, parallel, Ca2+-triggered vesicle-fusion reactions controlled by different synaptotagmins.
Olfactory bulb neurons were cultured from newborn mice as described (Cao et al., 2011). Briefly, olfactory bulbs were dissected from newborn wild-type mice of either sex (P1-P3), digested with papain (10 U/ml in 1 mM CaCl2 and 0.5 mM EGTA) for 20 min in an incubator, and dissociated with a 1 ml pipette. Dissociated olfactory bulb cells were plated on Matrigel-coated circular glass cover slips (diameter = 11 mm) in 24 well dishes, and cultured for 14-16 days in vitro (DIV) in 1 ml of MEM (GIBCO) supplemented with B27 (GIBCO), glucose, transferrin, fetal bovine serum and Ara-C (Sigma).
All lentiviral complexin KD and rescue constructs employed here were described previously (Maximov et al., 2009; Yang et al., 2010), except for the complexin KD/rescue constructs used for the pHluorin-Syt10 imaging experiments in which the IRES-GFP was deleted from the standard plasmids. The pHluorin-Syt10 construct was also described earlier (Cao et al., 2011). In the myc-Syt10 construct, the myc-epitope sequence was fused to the N-terminus of Syt10 without a linker or a signal peptide. In the IGF-1-Flag construct used, the Flag tag was inserted immediately after the IGF-1 signal peptide (Pfeffer et al, 2009). In the ANF-Venus construct (Burke et al, 1997; obtained from Dr. Weiping Han), the cDNA encoding the wild-type atrial natriuretic peptide A precursor is C-terminally fused via a linker (sequence: GGGCCCGGGGGATCCACC) to mVenus. In the phogrin-tdTomato or pHluorin-phogrin constructs (gift of Dr. Weiping Han), the cDNA encoding wild-type Phogrin is C-terminally fused via a linker (sequence: GGACCGGTCGCCACC) to tdTomato, of pHluorin is inserted in frame into the KpnI site of the phogrin extracellular coding sequence. All constructs were cloned into lentiviral expression vectors, and lentiviruses were produced essentially as described (Pang et al., 2010; Maximov et al., 2009). The lentiviral expression vector (control vector and shRNA-expressing vectors) and three helper plasmids (pRSV–REV, pMDLg/ pRRE, and vesicular stomatitis virus G protein expressing plasmid) were co-transfected into human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection) at 4, 2, 2, and 2 μg of DNA per 25 cm2 culture area, respectively. At 48 h after transfection, the HEK293 cell culture medium was collected and clarified by centrifugation (500 g for 5 min), and the supernatant containing lentiviral particles was added directly to the medium of cultured olfactory bulb neurons maintained in 24-well plates. For all experiments, olfactory bulb neurons were infected on DIV 2-3, and used for electrophysiological analyses on DIV14-16, or for IGF-1 secretion measurement on DIV7-DIV8.
Electrophysiological analyses were performed in cultured olfactory bulb neurons on DIV14-16 essentially as described (Cao et al, 2011). The bath solution contained (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES and 10 Glucose, adjusted to pH7.4 with NaOH. The whole-cell intracellular pipette solution contained (in mM): 135 CsCl, 10 HEPES, 1 EGTA, 1 Na-GTP, 4 Mg-ATP and 10 QX-314, adjusted to pH7.4 with CsOH. Patch pipettes were pulled from borosilicate glass capillary tubes (Warner Instruments, Cat #64-0793) using a PC-10 pipette puller (Narishiage Inc.). The resistance of pipettes filled with intracellular pipette solution varied between 3.0-3.5 MΩ. Presumptive mitral/tufted and granule/periglomerular neurons were identified by microscopy by their size, shape and visibility of their nuclei (Cheng and Gong, 2009). Mitral neurons were patched and kept in voltage-clamp (at -70mV) whole-cell recording modes using a MultiClamp 700B amplifier (Molecular Devices, Inc.). After establishment of the whole-cell configuration and equilibration of the intracellular pipette solution with the cytoplasm, the series resistance was compensated to 10-15 MΩ. Recordings with series resistances of >15 MΩ were rejected. Membrane capacitance (Cm) and input resistance (Rm) were measured using Clampex 10 (Molecular Devices, Inc.). In brief, the Membrane Test Module in Clampex 10 generates a 5 mV square wave voltage step (30 ms) in voltage-clamp mode. The Cm is automatically calculated by Clampex 10 based on the waveform of the resulting current transients (e.g. Gentet et al 2000). For the details, see pClamp 10 User Guide page 163-166. Evoked synaptic responses were triggered using a standard stimulation current (90 μA, 1 ms) with a bipolar electrode (FHC, CBAEC75 Concentric Bipolar Electrode OP: 125 mm SS; IP: 25 mm Pt/lr) placed at a distance of 100-150 μm from the soma of recorded neurons. The frequency, duration, and magnitude of the extracellular stimulus were controlled with a Model 2100 Isolated Pulse Stimulator (A-M Systems, Inc.) synchronized with Clampex 10 data acquisition software (Molecular Devices, Inc.). Excitatory postsynaptic currents were pharmacologically isolated by adding GABAa receptor blocker picrotoxin (50 μM) to the extracellular bath solution. mEPSCs were monitored in the presence of tetrodotoxin (TTX, 1 μM) to block action potentials. Data were analyzed using Clampfit 10 (Molecular Devices, Inc) and Origin 6.0 (Microcal Software Inc.).
For all experiments, the IGF-1 concentration in the medium was determined with the Quantikine Mouse/Rat IGF-1 ELISA (MG100, R&D Systems, Inc.), using serial dilutions of a mouse IGF-1 stock solution (2 μg/l) to generate a standard curve. All IGF-1 secretion experiments were performed at DIV7~8 with 1 h incubations. Under standard conditions, secretion was stimulated by incubations in either 5 mM KCl (control, representing regular culture medium) or 15 mM KCl (depolarizing medium) in the presence of 1 μM TTX; the osmolarity of the 5 mM KCl medium was adjusted with NaCl to that of the 15 mM KCl medium. For Ca2+-titration experiments of IGF-1 secretion, neuronal culture medium containing different Ca2+-concentrations was prepared from MEM without calcium (M8167, Sigma Aldrich) by adding appropriate concentrations of CaCl2, with all other components being identical to those used for regular experiments. To rule out the possibility that synaptic transmission activated by the increase in KCl may contribute to stimulating IGF-1 secretion, the effect of additions of blockers of major neurotransmitter receptors (20 μM CNQX, 50 μM APV, 0.1 mM MCPG and 50 μM picrotoxin) were analyzed (Fig. 3B). To exclude the possibility of unspecified artifacts due to the K+ stimulation, secretion was also stimulated by inducing membrane depolarization (in the absence of TTX) by field stimulation using a pair of parallel platinum electrodes (distance ~10 mm), and a 10 Hz 1 min stimulus train of 100 μA pulses (Fig. 5C). The position of electrodes was carefully adjusted with micromanipulator to be 50-100 μm above the neuronal culture in the recording chamber which contained 400 μl extracellular solution. In preliminary experiments, the effectiveness of field stimulation configuration was validated by simultaneous whole-cell recordings. To avoid possible interference by synaptic transmission, synaptic receptor blockers (20 μM CNQX, 50 μM APV, 100 μM MCPG and 50 μM picrotoxin) were added to the extracellular solution. The culture medium was collected 1 hr after the field stimulation for IGF-1 concentration measurements. Finally, to exclude any possible role for a non-Ca2+-stimulus in IGF-1 secretion, we permeabilized cultured olfactory bulb neurons using digitonin as described (Grabner and Fox, 2006). Wild type cultured olfactory bulb neurons on coverslips were positioned in wells of 24-well culture plates. The perfusion solution covering the coverslip (250 ul) could be rapidly changed with coordinated suction (vacuum) and addition (pipetman) of the perfusion solution. With this experimental paradigm, the neuronal cultures were sequentially treated with 1) perfusion solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES-NaOH pH 7.4, 2 mM MgCl2, 1 mM Na2ATP, 0 mM CaCl2, 100 μM EGTA) for 2 min, then 2) the same perfusion solution with 25 μM digitonin for 10 seconds, then 3) the same perfusion solution without digitonin but with different free Ca2+-concentrations (0 μM, 1 μM, 10 μM and 100 μM) for 30 min. In addition, receptor blockers (20 μM CNQX, 50 μM APV, 100 μM MCPG and 50 μM picrotoxin) and TTX (1 μM) were present in the perfusion solution. The relationship between free and total Ca2+-concentrations was calculated using the online calculation tool at Stanford University (http://www.stanford.edu/~cpatton/CaEGTA-TS.htm). With the perfusion solution used in the experiments (25 °C temperature, pH 7.4, ionic strength of 0.163, and 0.1 mM EGTA), 100, 10, and 1 μM free Ca2+ correspond to 200, 109, and 90 μM total Ca2+, respectively. After treatments, the perfusion solution was collected for IGF-1 measurements (Fig. 5D).
Wild-type cultured olfactory bulb neurons at DIV3 were either single-infected or super-infected with a combination of two lentiviruses encoding pHluorin-Syt10 (Cao et al, 2011), myc-Syt10, IGF1-Flag, phogrin-tdTomato and ANF-Venus (Burke et al, 1997). For immunocytochemistry, cultured olfactory bulb neurons were rinsed with PBS, and fixed with 4% PFA for 20 min in room temperature, blocked in 4% goat serum in PBS for 60 min, then incubated with primary antibodies (50 μl) in a damp box for two hour at room temperature. The following primary antibodies were used for immunocytochemistry: polyclonal rabbit anti-Synapsin (E028); monoclonal mouse anti-GFP (Clontech, JL-8); monoclonal and polyclonal rabbit anti-Flag (Sigma Aldrich); Guinea pig anti- Vesicular Glutamate Transporter 1 (VGluT1, Synaptic Systems); polyclonal rabbit anti-Myc (Sigma Aldrich); polyclonal rabbit anti-mCherry (Synaptic Systems). Coverslips were rinsed 3x with PBS for 10 min, incubated with fluorescent secondary antibodies (1:500, Alexa 488-, 546-, or 633-labeled highly cross-absorbed goat anti-mouse, goat anti-rabbit and goat anti-guinea pig antisera, Molecular Probes Inc.) for 60 min at room temperatures, washed, and mounted with mounting medium (Sigma Aldrich). Images were collected with a Leica TCS2 confocal microscope with a 63x objective. Samples were excited by 488 nm, 543 nm or 633 lasers in sequential acquisition mode to avoid signal leaking. Identical acquisition settings were applied to all samples of an experiment. Saturation was avoided by monitoring pixel intensity with glowing mode. Confocal images were sequentially recorded with increasing zooms. Normally, a 1x zoom was used to acquire a global view of the neurons; a 3x zoom was used to visualize detailed dendritic and axonal structures; and a 5x zoom was used to visualize the detailed puncta structure within neuronal processes. Co-localization analyses were done using Image J with the plug-in JACoP (http://rsbweb.nih.gov/ij/plugins/track/jacop.html). Only pictures with a 5x zoom with detailed puncta structure were used for co-localization analyses. Although the JACoP plug-in can automatically calculate the Pearson's Coefficient (PC), the raw PC needs to be corrected by subtracting the background PC (Bolte and Cordelieres, 2006), which was obtained by translating one of the images for 15 pixels in both directions. The pictures used for measuring puncta sizes were acquired with a 63x objective with optical zooming of ~5, which resulted in image dimensions of ~50 μm. The diameter was analyzed with Image J. In brief, the scale of the pictures was set in Image J based on the physical dimension of the picture recorded by the Leica Confocal System. After converting the pictures from RGB color mode to 16-bit mode, the puncta in the pictures were binarized and measured automatically by Image J. To plot the cumulative distribution curves, 300~400 puncta for each label were used. Each pair of distribution curves was tested statistically with the Kolmogorov–Smirnov test.
Cultured olfactory bulb neurons co-infected with pHluorin-Syt10 and complexin KD lentiviruses at DIV2-3 were imaged at DIV14-16. Chambers containing a coverslip with cultured neurons were continuously perfused at 5 ml/min with Tyrode's solution (140 mM NaCl, 5 mM KCl, 5.5 mM glucose, 2 mM CaCl2 and 2 mM MgCl2 and 20 mM HEPES-NaOH pH 7.4). Imaging experiments started with perfusing Tyrode's solution containing 50 mM NH4Cl and 90 mM NaCl instead of 140 mM NaCl to visualize all pHluorin-Syt10 containing vesicles in a mitral/tufted neuron. After washout of NH4Cl by perfusion of regular Tyrode's solution, the chamber was superfused with depolarizing Tyrode's solution containing 15 mM KCl and 130 mM NaCl instead of 5 mM KCl and 140 mM NaCl, followed again by a washout superfusion period with regular Tyrode's solution. Mitral cells were imaged with a water immersion objective (40X) in a Zeiss confocal imaging system at room temperature. Sequential time-lapse images (512×512) acquired at 4 s intervals were used to analyze the dynamics of pHluorin-Syt10 in response to NH4Cl and membrane depolarization. The image analysis for pHluorin-Syt10 live-cell imaging was done with Zeiss Software ZEN. A 4 pixel × 4 pixel region of interest (ROI) was centered on individual pHluorin-Syt10 puncta in the raw images. The built-in measurement module in ZEN software automatically measured the averaged fluorescence intensity within ROI as a function of time. The exocytosis of a pHluorin-Syt10 puncta was quantified by the change of fluorescence (ΔF) before and after solution perfusion divided by the initial fluorescence (F0), in which F0 is the average fluorescence in the first four frames before onset of stimulus. The image analysis did not involve any thresholding. The averaged fluorescence intensity of an ROI would be completely distorted after thresholding because it would make the pixels on the images either black (intensity=0) or white (intensity=255).
All experiments were performed in a ‘blinded’ fashion, i.e., the experimenter was unaware of the precise manipulation a sample had been subjected to. At least three independent cultures for each type of experiment were performed. Statistical comparisons were made using Student's t-test or two-way ANOVA as indicated.
The selective requirement for Syt10 but not Syt1 in IGF-1 exocytosis suggested that IGF-1 vesicles containing Syt10 (Cao et al., 2011) are distinct from LDCVs containing Syt1 (Walch-Solimena et al., 1993). However, the neuropeptide-like structure and Ca2+-dependent secretion of IGF-1 by olfactory bulb neurons cast doubt on this notion, as it would suggest the unexpected conclusion that a neuron can express two different types of peptidergic secretory vesicles. Thus, we examined whether markers for LDCVs and for IGF-1-containing vesicles localize to the same or different vesicles in cultured olfactory bulb neurons. In these neurons, we primarily analyzed glutamatergic mitral and tufted neurons that can be easily distinguished from the significantly smaller GABAergic granule and periglomerular neurons. As markers for LDCVs, we employed Syt1, the neuropeptide ANF, and the intrinsic membrane protein phogrin (Zahn et al., 2001; Wasmaier et al., 2005), and as markers for IGF-1-containing vesicles we employed Syt10 and IGF-1 (Cao et al., 2011).
We first expressed tagged phogrin in cultured olfactory bulb neurons via a lentivirus, and analyzed the relative distributions of endogenous Syt1 with respect to phogrin. We observed a complete co-localization of Syt1 with phogrin in the cell body of the neurons (Fig. 1A), whereas presynaptic terminals contained only Syt1 but not phogrin (not shown). Next, we compared the relative localizations of lentivirally expressed, tagged phogrin to that of ANF, Syt10, and IGF-1, or of the endogenous synaptic markers vGlut1 or synapsin. The vesicular localization of ANF overlapped with that of phogrin in both the neuronal soma (Fig.1B) and their dendrites (Fig.2E). The synaptic vesicle marker vGlut1 was not observed in somatic vesicles in the neurons (Fig.1C), nor was synapsin co-localized with phogrin vesicles in dendrite (Fig.2H). Importantly, like phogrin we found that Syt10 and IGF-1 were also present in somatic (Figs.1D and 1E) and dendritic (Figs. 2B and 2G) vesicles that, however, were different from the vesicles containing phogrin.
The phogrin localization experiments suggest that ANF-containing LDCVs also contain Syt1, but that IGF-1 and Syt10 are present on separate vesicles. To confirm this conclusion, we compared the relative localization of Syt10 with that of IGF-1, ANF, and synapsin. We detected a complete overlap in the localizations of Syt10 and IGF-1 (Fig.1F and and2A),2A), but no overlap beween Syt10 and ANF (Fig.1G and and2C)2C) or synapsin (which is specific for synaptic vesicles; Figs. 1H; Cao et al., 2011). Accordingly, we also found no overlap in the localizations of IGF-1 and either ANF (Fig.1I and Fig.2F) or synapsin (Figs. 1J), or of ANF and synapsin (Fig. 1K and Fig.2D). The relative degree of co-localization of different markers in independent experiments was quantified using Pearson's coefficient, unequivocally demonstrating that the soma of cultured olfactory bulb neurons contains two different classes of peptidergic vesicles (Fig. 1L). As assayed by light microscopy, ANF- and phogrin-containing LDCVs are significantly smaller than Syt10- and IGF-1-containing vesicles (Fig. 1M). Thus, olfactory bulb neurons produce two separate classes of peptidergic secretory vesicles, LDCVs containing Syt1 and even larger secretory vesicles containing Syt10 and IGF-1 (Fig. 2I).
Since the role of complexin in synaptic vesicle exocytosis and neurotransmitter release has not previously been investigated in olfactory bulb neurons, we examined the role of complexin in synaptic vesicle exocytosis in olfactory bulb neurons using complexin knockdown (KD) experiments. As described previously for cortical neurons (Maximov et al., 2009), we infected cultured olfactory bulb neurons with lentiviruses encoding either only EGFP (Control), EGFP together with shRNAs suppressing complexin-1 and -2 expression (Cpx KD), or EGFP together with the complexin shRNAs and various rescue proteins.
Recordings of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in mitral/tufted neurons revealed that, as in cortical neurons (Maximov et al., 2009; Yang et al., 2010), the complexin KD produced a >2-fold increase in mEPSC frequency without significantly altering the mEPSC amplitude (Figs. 3A and 3B). This phenotype was rescued by wild-type but not ‘4M-mutant’ complexin that is unable to bind to SNARE complexes (Tang et al., 2006; Maximov et al., 2009), demonstrating that complexin function requires SNARE-complex binding. Interestingly, the complexin KD additionally decreased the capacitance and increased the input resistance of mitral neurons; again, this phenotype was rescued by wild-type but not 4M-mutant complexin (Fig. 3C). It is notable that the latter complexin KD phenotype differs from that of cultured cortical neurons in which the complexin KD produces no change in capacitance or input resistance (Maximov et al., 2009; Yang et al., 2010), but resembles the phenotype of the Syt10 KO (Cao et al., 2011). The capacitance and input resistance increases and decreases with neuronal development, respectively. IGF1 is a growth factor that promotes neuronal development, such as cell body growth and dendritic arborization. Therefore, capacitance and input resistance were used as proxy for IGF-1 secretion (Cao et al, 2011).
Next, we monitored the effect of the complexin KD on evoked EPSCs which we measured in the presence of increasing concentrations of extracellular Ca2+. As described previously in cortical neurons (Yang et al., 2010), the complexin KD produced a ~3-fold decrease in neurotransmitter release in olfactory bulb neurons as assessed by the EPSC amplitude (Figs. 4A and 4B). Fitting the EPSC amplitude data obtained at different Ca2+-concentrations to a Hill function (Fernandez-Chacon et al., 2001) showed that the complexin KD caused a small but significant increase in the apparent Ca2+-affinity and a corresponding decrease in the apparent Ca2+-cooperativity of release (Fig. 4C and 4D), again consistent with earlier data (Yang et al., 2010). Analysis of the Ca2+-dependence of the EPSC amplitude using a different independent method pioneered by Dodge and Rahamimoff (1967) revealed a similar Ca2+-cooperativity of release in control neurons as the Hill function fittings, but an even larger decrease in the apparent Ca2+-cooperativity of release in complexin KD neurons (Figs. 4E and 4F). Although these analyses do not allow an assessment of the absolute Ca2+-cooperativity of release because they depend on titrations of extracellular Ca2+, they do confirm earlier conclusions (Yang et al., 2010) that the complexin KD not only impairs neurotransmitter release, but also decreases the apparent Ca2+-cooperativity of neurotransmitter release.
The KD of complexins alters the capacitance and input resistance in olfactory bulb neurons but not in cortical neurons, and no such changes in capacitance and input resistance are observed after deletion or KD of Syt1. The changes in capacitance and input resistance in complexin KD neurons mimic the changes produced by deletion of Syt10 which impairs IGF-1 secretion, thereby leading to a decrease in neuronal size and an increase in electrical resistance (Cao et al., 2011). To test whether the changes in capacitance and input resistance in complexin KD neurons are produced by the same mechanism, we examined the effect of the complexin KD on IGF-1 secretion. We stimulated cultured olfactory bulb neurons by mild depolarization with 15 mM K+ in the presence of tetrodotoxin (to block action-potentials), and measured the concentration of secreted IGF-1 in the medium. Depolarization of olfactory bulb neurons by 15 mM K+ in Ca2+-containing medium induced a large increase in IGF-1 secretion into the medium (Fig. 5A). In contrast, the same depolarization of olfactory bulb neurons in Ca2+-free medium produced no statistically significant increase in IGF-1 secretion, although there was a small non-significant increase (Fig. 5A, P>0.05). IGF-1 secretion was not altered by addition of receptor antagonists blocking all neurotransmitter signaling (Fig. 5A), suggesting that 15 mM K+ stimulated IGF-1 secretion directly via depolarization-induced Ca2+-influx, and not indirectly via increased synaptic activity.
The complexin KD significantly reduced Ca2+-dependent IGF-1 secretion, but not baseline IGF-1 secretion (Fig. 5B). As before, this phenotype was rescued by wild-type but not by 4M-mutant complexin. Similar to synaptic vesicle exocytosis, the complexin KD only partly blocked Ca2+-induced IGF-1 exocytosis, possibly because the KD efficiency is not 100%, or because complexin is not as essential for Ca2+-triggering of exocytosis as Syt1 (for synaptic vesicle exocytosis) or as Syt10 (for IGF-1 exocytosis).
The impairment in IGF-1 secretion induced by the complexin KD was independent of the stimulation method used. We observed the same impairment of IGF-1 secretion when we stimulated olfactory bulb neurons electrically by triggering trains of action potentials, which were again elicited in the presence of neurotransmitter receptor blockers to occlude any possible interference produced by synaptic transmission (Fig. 5C). Moreover, to confirm with yet another independent assay that Ca2+ directly induced IGF-1 exocytosis, we assayed IGF-1 exocytosis in cultured olfactory bulb neurons that had been permeabilized with digitonin (Grabner and Fox, 2006). We measured IGF-1 secretion induced by addition of Ca2+ to the permeabilized neurons (Fig. 5D). Again, we observed Ca2+-dependent IGF-1 exocytosis that was partially blocked by the complexin KD. Thus, Ca2+ directly triggers IGF-1 exocytosis – presumably by binding to Syt10 (Cao et al., 2011) – and complexin is essential for this Ca2+-triggered IGF-1 exocytosis independent of synaptic transmission.
We then asked whether IGF-1 secretion and neurotransmitter release exhibit a similar Ca2+-dependence in olfactory bulb neurons, and whether the complexin KD alters the Ca2+-dependence of these secretory processes in a similar manner. We found that the Ca2+-dependence of IGF-1 secretion and synaptic vesicle exocytosis in olfactory bulb neurons were similar, and that the complexin KD produced an almost identical effect on Ca2+-dependent IGF-1 exocytosis as on neurotransmitter release (Figs. 6A and 6B). Direct comparison of the Ca2+-concentration dependence of EPSCs and IGF-1 secretion failed to reveal significant differences at intermediate and higher Ca2+-concentrations (Fig. 6C), consistent with the similar Ca2+-affinities of Syt1 and Syt10 (Sugita et al. 2002). It is possible that the relative amounts of neurotransmitter and IGF-1 release differ at low Ca2+-concentrations, but it is difficult to accurately test this possibility because of the low sensitivity of IGF-1 secretion assay which renders precise measurements of low levels of IGF-1 secretion difficult.
To further analyze the impairment in IGF-1 secretion we observed after the complexin KD, we used imaging and directly probed the effect of the complexin KD on Syt10-containing vesicles. We lentivirally expressed pHluorin-tagged Syt10 in cultured olfactory bulb neurons, and monitored depolarization-induced exocytosis of Syt10-containing vesicles with fluorescence imaging (Cao et al., 2011). In these experiments, we first exposed the neurons to 50 mM NH4Cl to visualize pHluorin-Syt10 containing vesicles, washed out the NH4Cl, exposed the neurons to 15 mM K+ to stimulate Ca2+-induced exocytosis, and finally washed out the 15 M K+ solution. All experiments were performed in the presence of 1 μM tetrodotoxin to suppress depolarization-induced network activity that may indirectly induce exocytosis of pHluorin-Syt10 containing vesicles. Using this method, we visualized Syt10-containing vesicles in dendrites, and monitored their depolarization-induced exocytosis under control conditions and after complexin KD without or with rescue (Fig. 7).
Small elevations in the extracellular K+-concentration (from 5 to 15 mM) rapidly induced Ca2+-dependent exocytosis of Syt10-containing vesicles in control neurons (Figs. 7A-7B and Fig.8A-8B), but were more than 3-fold less effective in complexin KD neurons (Figs. 7C-7D and Figs.8A-8B). Although some rapid exocytosis of Syt10-containing vesicles was still observed in complexin KD neurons, the complexin KD decreased the overall rate of exocytosis. In contrast, the complexin KD had no effect on the strength of the pHluorin-Syt10 signal produced by NH4Cl (Figs. 7A-D and Figs. 8A-8B). The phenotype of the complexin KD was fully rescued by wild-type complexin (Figs. 7E-7F and Figs. 8A-8B), demonstrating that the complexin KD specifically impaired exocytosis and not vesicle biogenesis. Thus, complexin is directly required for Ca2+-triggered exocytosis of Syt10-containing secretory vesicles in olfactory bulb neurons.
In synaptic exocytosis, complexin functions both as an activator and as a clamp (e.g., see Reim et al., 2001; Tang et al., 2006; Huntwork and Littleton, 2007; Xue et al., 2007, 2009, and 2010; Cai et al., 2008; Maximov et al., 2009; Strenzke et al., 2009; Yang et al., 2010; Cho et al., 2010; Hobson et al., 2011; Martin et al., 2011). These functions require distinct complexin sequences. To test which of these complexin functions operate in IGF-1 secretion, we performed rescue experiments with mutants of complexin that selectively disrupt these functions, namely a deletion of the N-terminal 26 complexin residues that blocks its activating but not its clamping function (Maximov et al., 2009), and the ‘poorclamp’ point mutation in the accessory α-helix that blocks its clamping but not its activating function (Yang et al., 2010). In addition, we analyzed a mutant that disrupts both complexin functions (the deletion of the N-terminal 40 complexin residues), and a mutant designed to enhance clamping (the ‘superclamp’ mutant; Giraudo et al., 2009). The poorclamp mutant was generated in complexin-1 by combined substitution of three residues (K26E/L41K/E47K) that were mutated because they align with the synaptobrevin-2 SNARE-motif sequence in a reverse orientation (Fig. 10A; Yang et al., 2010), suggesting that these residues may promote insertion of the accessory complexin α-helix into the SNARE complex during clamping (Giraudo et al. 2009). Mutating these residues was thus predicted to decrease the clamping function of complexin, as confirmed in cultured cortical neurons (Yang et al. 2010).
All of the complexin mutants tested (Figs. 9A and 10A) altered neurotransmitter release in olfactory bulb neurons in a manner similar to their previously observed effects in cortical neurons (Figs. 9B-9C, 10B, and 10C; Yang et al., 2010). Interestingly, the complexin poorclamp mutant not only failed to rescue the increased mEPSC frequency in complexin KD neurons, it even aggravated this increase in a statistically significant fashion (Fig. 10C). At the same time, the poorclamp mutant rescued the decrease in evoked release in complexin KD neurons (Yang et al., 2010). The increased mini frequency in neurons expressing the poorclamp complexin mutant could be explained by a dominant negative effect, or by rescue of priming (which is impaired by the complexin KD; see Yang et al., 2010) without a change in clamping from the regular KD condition. Increased priming may render more vesicles ready for spontaneous release, and thus indirectly increase the mini rate. In looking back, we previously observed a similar effect of the poorclamp mutant (but not a significant difference) on the mini frequency in cultured cortical neurons (Yang et al., 2011). Cultured olfactory bulb neurons may be more reliable than cultured cortical neurons in revealing this phenotype because it is easier in cultured olfactory bulb neurons to record from specific neuron types (i.e., mitral and tufted neurons).
We next measured the effect of the various complexin mutations on Ca2+-stimulated IGF-1 secretion from olfactory bulb neurons. Using capacitance and input resistance measurements as a proxy for IGF-1 secretion or employing direct IGF-1 secretion measurements, we found that the clamping mutants did not change the baseline level of IGF-1 secretion under control conditions, nor did they later evoked IGF-1 secretion (Fig. 10). This result indicates that complexin-clamped “spontaneous” IGF-1 release does not provide a major contribution to IGF-1 secretion, and highlights the greater importance of the activation function of complexin. Indeed, the activation mutants of complexin did not rescue the IGF-1 secretion phenotype (Figs. 9D-9E and 10D-10E), revealing that complexin function in IGF-1 exocytosis completely relies on its role as an activator. It would have been interesting to measure the Ca2+-dependence of IGF-1 secretion with the N-terminal complexin mutants that block its activation function. However, the large decrease in IGF-1 secretion caused by these mutations and the low sensitivity of IGF-1 measurements compared to EPSC measurements make it difficult to accurately determine the Ca2+-dependence of the little bit of IGF-1 secretion that is left with these complexin mutants.
The present study asks two fundamental questions. First, does the Ca2+-dependent exocytosis of IGF-1 we recently described in olfactory bulb neurons (Cao et al., 2011) represent a standard pathway of neuropeptide secretion similar to the well-characterized pathway of LDCV exocytosis (Winkler and Fischer-Colbrie, 1998), or does it constitute a novel distinct secretory pathway? Second, is the triggering of IGF-1 secretion mediated by Ca2+-binding to Syt10 (but not to the synaptotagmins normally implicated in neuropeptide secretion) dependent on complexins similar to neurotransmitter secretion and chromaffin granule exocytosis induced by Ca2+-binding to Syt1, Syt2, Syt7, or Syt9?
Our data show that the vesicles mediating IGF-1 secretion are distinct in location and size from those mediating neuropeptide secretion (Figs. 1 and and2),2), but that the exocytosis of these IGF-1-containing vesicles is nevertheless dependent on complexins (Figs. 3--10).10). Moreover, our data demonstrate that complexin functions in IGF-1 secretion as an activator that enables rapid Ca2+-triggered exocytosis of IGF-1 containing vesicles. Thus, olfactory bulb neurons (most likely mitral neurons) generate two classes of peptidergic secretory vesicles whose Ca2+-triggered exocytosis depends on different isoforms of synaptotagmins but nevertheless uniformly requires complexins.
The evidence for our conclusions is as follows:
Based on these data, we conclude that olfactory bulb neurons contain at least two independent and distinct pathways of Ca2+-regulated polypeptide secretion – a previously characterized LDCV pathway for neuropeptide secretion utilizing Syt1 as a Ca2+-sensor (Walch-Solimena et al., 1993; Zhu et al., 2007), and a novel pathway for IGF-1 secretion using Syt10 as a Ca2+-sensor (Cao et al., 2011). Both pathways are dependent on complexins, despite the fact that Syt1 and Syt10 are functionally distinct and non-redundant (Cao et al., 2011). Thus, complexins may generally cooperate with synaptotagmins in exocytosis, and not only function as co-factors for Syt1-dependent exocytosis in synaptic and chromaffin exocytosis (Reim et al., 2001; Cai et al., 2008; Xue et al., 2007; Maximov et al., 2009), suggesting a much broader role in exocytosis than previously envisioned. Thus, the present data indicate that the phenotype of genetic complexin manipulations in mice, flies, and worms may be due to a general if partial impairment of multiple types of exocytosis. The general role of complexins in exocytosis is likely to be primarily as a positive activator of SNARE complexes prior to fusion-pore opening since this function appears to be more essential than the clamping function of complexin in synaptic vesicle exocytosis (Yang et al., 2010) and IGF-1 exocytosis (Fig. 9 and and10).10). It should be noted, however, that complexin – different from synaptotagmin – does not seem to be absolutely required for neurotransmitter release or IGF-1 exocytosis, even when analyzed in double complexin-1/2 KO neurons (Reim et al., 2000). Thus, complexin acts more as a co-factor for synaptotagmins that improves the secretory reaction than as a building block of the secretory machinery.
Interestingly, the clamping activity of complexin seems to have no significant role in IGF-1 secretion even though it appears to exert a significant control of neurotransmitter release at synapses. It is possible that the clamping function of complexin does not play a major role in controlling IGF-1 exocytosis, or that the rate of spontaneous IGF-1 exocytosis is too small to allow detection of spontaneous IGF-1 release events even after unclamping. The sensitivity of IGF-1 ELISA assays is much lower than that of mEPSC measurements which allow detection of single vesicle exocytosis events, strongly supporting the second hypothesis.
Our results complement recent findings that complexin is required for postsynaptic AMPA-receptor exocytosis triggered by LTP, and suggest that postsynaptic regulated exocytosis may also be dependent on a synaptotagmin (Ahmad et al., 2012). Furthermore, given the fact that complexin and at least Syt14, Syt15, and Syt16 appear to be universally expressed in all cells at low levels (McMahon et al., 1995; Fukuda, 2003a and 2003b; Herrero-Turrion et al., 2006), our results suggest that a complexin/synaptotagmin-dependent pathway may operate in all cells to mediate an as yet unidentified type of exocytosis that could be regulated by a signal different from Ca2+. The universal role for complexin in exocytosis thus emerging is consistent with the evolutionary conservation of complexins and synaptotagmins in all animals including sponges, and suggests that manipulating complexin function may be a general approach to influence regulated exocytosis in cells.
Why do olfactory bulb neurons develop a specific, activity-dependent pathway of IGF-1 exocytosis? A first clue to this question came from studies demonstrating that IGF-1 performs an essential role in the continuous activity-dependent assembly of the neural circuitry of the olfactory bulb, which needs to be re-wired throughout life because of adult neurogenesis of granule cells (Scolnick et al., 2008; Hurtado-Chong et al., 2009; Vicario-Abejon et al., 2003; Pixley et al., 1998). Moreover, a recent study revealed that neuronal diversity in the olfactory bulb may in part result from a neuron's activity-dependent adaptation to the local neural circuits (Angelo et al., 2012). However, the underlying molecular mechanisms are unclear. Our results may explain how activity adjusts the properties of the neurons in the circuitry of the olfactory bulb. In the olfactory bulb, IGF-1 secreted from a mitral cell may, in a paracrine manner, only activate adjacent mitral cells sharing an identical glomerulus while leaving other, more distant, mitral cells unaffected. Since IGF-1 has been widely implicated in a variety of neuronal and circuit functions (Blair and Marshall 1997; Ramsey et al., 2005; Xing et al., 2007; Man et al., 2000; Wang et al., 2000; Kim et al., 2007; Tropea et al., 2006), our data suggest that the Ca2+-triggered IGF-1 exocytosis mediated by complexins and synaptotagmin may be involved the regulation of mitral cell diversity in olfactory bulb. Moreover, similar mechanisms could potentially operate in other brain areas, linking activity-dependent exocytosis to the development of specific neuronal properties and neural circuits.
We thank Dr. Yea Jin Kaeser-Woo for advice and provision of plasmids. This paper was supported by NIH grants P50 MH086403 and R01 MH089054 (to T.C.S.).
Author Contribution: T.C.S., P.C. and X.Y. designed research; P.C. and X.Y. performed research; P.C. and X.Y. analyzed data; T.C.S. and P.C. wrote the paper.
Conflict of interest: The authors declare no competing financial interests.