Method Details

The following primary antibodies were used in this study: Mouse antibodies against: CtBP1 (immunocytochemistry (ICC) 1:1,000, western blotting (WB) 1:5,000, BD Biosciences, 612042), CtBP2 (WB 1:2000 BD Biosciences, 612044) synaptotagmin1 lumenal domain Oyster 550 or CypHer5E-labeled (ICC 1:200, Synaptic Systems, 105311 and 105311CpH), rab5 (ICC 1:500, Synaptic Systems, cells stained with this antibody were fixed with ice-cold methanol for 10 min, followed by rehydration in PBS for 20 min, 108011), rab7 (ICC 1:1,000, Abcam, ab50533), phosphoserine/threonine (WB 1:1000, BD Biosciences, 612548), GluA Oyster 550-labeled (ICC 1:200, Synaptic Systems, 182411 C3), α-tubulin (WB 1:1000, Sigma Aldrich); Rabbit antibodies against: CtBP1 (ICC 1:1,000, WB 1:1,000, Synaptic Systems, 222002), GFP (ICC 1:1,000, WB 1:5,000, Abcam, ab 6556), SV2B (ICC 1:200, Synaptic Systems, 119103), GAPDH (WB 1:3000, Abcam, ab37168), synaptotagmin1 lumenal domain Oyster 550-labeled (ICC 1:200, Synaptic Systems, 105103C3), synaptotagmin 1 lumenal domain (WB 1:1000, Synaptic Systems, 105102), dynamin1 (ICC 1:1000, Abcam, ab3456), rab22a (ICC 1:1000, Abcam, ab137093), Phospholipase D (WB 1:1000, Cell Signaling technologies, 3832S), Homer1 (ICC 1:500, Synaptic Systems, 160003); Guinea pig antibodies against: synapsin 1, 2 (ICC 1:1,000, Synaptic Systems, 106004), synaptophysin 1 (ICC 1:1,000, Synaptic Systems, 101004), Piccolo (WB 1:2000, Dick et al., 2001).

The following secondary cross-adsorbed antibodies were used in this study: Alexa 488- (ICC: 1:1,000), Cy3-(ICC: 1:1,000), Cy5-(ICC: 1:2,000), Alexa 680- (WB 1:20,000) conjugated whole IgGs against mouse, rabbit and guinea pig were obtained from Invitrogen/Mol. Probes, IRDye 800CW (WB 1:20,000) and Atto 647N (1:500, 610-156-121 and 611-156-122) from Rockland and Abberior STAR 580 (1:100, 2-0002-005-1 and 2-0012-005-8) from Abberior GmbH.

EGFP-tagged CtBP1 was generated by cloning the sequence for CtBP1-S into pEGFPC vector. Subsequently, the DNA cassette containing EGFP-CtBP1 was shuttled into FUGW H1 lentiviral vector (Leal-Ortiz et al., 2008), replacing EGFP coding sequence. The shRNAs against CtBP1 and YFP-CtBP2(NLS)-CtBP1 constructs were reported previously (Ivanova et al., 2015, Verger et al., 2006). All point mutations, including the silent point mutations for the rescue experiments, were introduced by inverse PCR using primers containing the mutations and CtBP1-S coding sequence cloned in pBluescriptII SK-(AgilentTechnologies). The ratio:sypHy construct and syp mOrange2 used in this study were reported in Lazarevic et al., 2017, Rose et al., 2013, and Egashira et al. (2015), respectively. All constructs were verified by sequencing.

Organotypic hippocampal slice cultures from Ctbp1 KO and WT littermates were prepared at postnatal day 0 and were cryo-fixed after 4-5 weeks in vitro under cryo-protectant conditions (20% bovine serum albumin in culture medium) using the High Pressure Freezing device HPM100 (Leica), and cryo-substituted in Freeze Substitution Processor EM AFS2 (Leica) according to previously published protocols (Imig and Cooper, 2017, Imig et al., 2014). For 2D analyses of synaptic morphology, electron micrographs were acquired from 60 nm-thick plastic sections with a transmission electron microscope (Zeiss LEO 912-Omega) operating at 80 kV. For 3D electron tomographic analysis of docked SV, 200 nm-thick plastic sections were imaged in a JEM-2100 transmission electron microscope (JEOL) operating at 200 kV. SerialEM (Mastronarde, 2005) was used to acquire single-axis tilt series (−60°/-55° to ± 55°/ ± 60°; 1° increments) at 25,000 fold magnification with an Orius SC1000 camera (Gatan, Inc.). Tomograms reconstructed from tilt series using the IMOD package (Kremer et al., 1996) had a voxel size of x,y,z = 1.82 nm. Tomogram acquisition and analyses were performed blindly. Quantifications were done manually using ImageJ (National Institutes of Health). The smallest SV distances from the outer leaflet of the SV membrane to the inner leaflet of the AZ plasma membrane were measured using the straight line tool of the ImageJ software. Only SVs observed to be in physical contact at their midline with the presynaptic membrane were considered docked (0-2 nm distance). The mean SV diameter was calculated from the area of the SV measured at its midline to the outer leaflet of the SV membrane using the elliptical selection tool of ImageJ.

For illustrative purposes, images depicting tomographic sub-volumes represent an overlay of seven consecutive tomographic slices produced using the slicer tool of the 3dmod software of the IMOD software package to generate an approximately 13 nm thick sub-volume.

Quantitative real-time PCR was performed as described in Ivanova et al. (2015). Total RNA was extracted from primary cortical cultures (DIV16) superinfected on the day of plating with lentiviral particles driving the expression of scrambled, shRNA944 and YFP-CtBP2(NLS)-CtBP1, using RNeasy Plus Mini Kit (QIAGEN) and following the instructions of the manufacturer. The transcript levels of BDNF and Arc were analyzed by a customized version of Rat Synaptic Plasticity RT² Profiler PCR Array (QIAGEN). To calculate the expression of BDNF and Arc in relation to a reference gene we used ΔΔCP method. We used the ‘second derivative maximum analysis’ method, available in the software of Roche LightCycler480, to determine the crossing point (CP) of the PCR. The expression of lactate dehydrogenase A was used as a reference to calculate the relative mRNA levels of BDNF and Arc.

Cortical neurons with cell density 10 million per 75‐cm2 flask were superinfected with lentiviral particles, driving the expression of EGFP-CtBP1. Cells (DIV16) were lysed in 10mM Tris–HCl, 150mM NaCl, 2% SDS, 1% deoxycholate and 1% Triton X-100 containing complete protease inhibitors (Roche), and PhosStop (Roche) and co-immunoprecipitations were performed using MicroMACS anti‐GFP MicroBeads and MicroColumns (Miltenyi Biotec) according to the instructions from the manufacturer.

Crude synaptosomal fraction (P2) was prepared as follows: First, cell or mouse brain homogenates were prepared in HEPES-buffered sucrose (4 mM HEPES pH 7.4, 0.32 M sucrose) and centrifuged at 1000 x g for 10 min to pellet the nuclear fraction (P1). The supernatant was then centrifuged at 12000 g for 20 min to give the crude synaptosomal pellet (P2). The crude synaptosomal fraction (P2) was lysed in 10 mM Tris–HCl, 150mM NaCl, 2% SDS, 1% deoxycholate and 1% Triton X-100 containing complete protease inhibitors (Roche), and PhosStop (Roche) and further subjected to IP or western blotting.

Protein samples were separated on 5%–20% Tris-glycine gels, or 3.5%–8% Tris-acetate gels as described previously (Ivanova et al., 2015) or on 10% (Bio-Rad TGX-Stain free gels) and blotted onto Millipore Immobilon FL PVDF membranes by tank or semidry blotting. Immunodetection was performed on Odyssey Infrared Scanner (LI-COR). For the quantification of the immunoblots the integrated density (ID) of signals was measured using ImageJ by setting rectangular ROIs with identical size around or using Image Studio Software (LI-COR). Samples of each experimental group were always loaded and quantified on the same membrane. TCE total protein stain was used for normalization in Figure 1B. In Figure S2A, GAPDH or α-tubulin were used for normalization in homogenates and P2 fraction, respectively. The values for ID of CtBP1 (Figures 7A–7D) were normalized to the corresponding expression levels of the two proteins in each experimental group. The antibodies used for immunodetection and the molecular weight of the markers are indicated in the figures.

Immunostaining of neurons was performed as described in Lazarevic et al. (2011). For quantifications, identical antibodies solutions were used for all coverslips from the same experiment. For the co-localization analysis, neurons were silenced with APV and CNQX for 10 minutes, in order to minimize the effect of the ongoing activity on the variance between synapses and then stimulated with 200 AP at 40 Hz. Cells were fixed within 30 s after the end of stimulation.

Staining with synaptotagmin 1 antibody (Syt1 Ab uptake) was performed by incubating the cells with fluorescently-labeled primary antibody dissolved in extracellular solution, containing 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, and 25 mM HEPES, pH 7.4 for 30 min at 37°C (Lazarevic et al., 2011) before fixation. For the imaging with CypHer5E-labeled anti-synaptotagmin1 antibody, cells were incubated with the antibody diluted in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 18 mM NaHCO3, pH 7.4 for 2-3 hours at 37°C prior imaging.

Epifluorescence images were acquired on a Zeiss Axio Imager A2 microscope with Cool Snap EZ camera (Visitron Systems) controlled by VisiView (Visitron Systems GmbH) software.

Confocal images in Figure S3A were acquired on a Leica SP5 confocal microscope. The format of the images was 2048x2048 pixels display resolution, 8 bit dynamic range, for acquisition 63x objective, NA 1.40 and 2x optical zoom were used, which results in a voxel size of approximately 50 nm.

Dual-color STED images (1024x1024 pixels display resolution, 8 bit dynamic range) were acquired on a Leica TCS SP8-3X gated STED microscope using a HC APO CS2 100x objective, NA 1.40, and 5x optical zoom, corresponding to a voxel size of approximately 23 nm. 16 times line averaging was applied on frames acquired at a scan speed 600 Hz. The built-in pulsed white light laser of the setup was used to excite Abberior STAR 580 and Atto 647N at 561 nm and 650 nm, respectively. The detection was done at 580-620 nm for Abberior STAR 580 and 660-730 nm for Atto647N. Both dyes were depleted using a pulsed 775 nm depletion laser. Time-gated detection of 0.5-1 ns to 6 ns was set for both STED channels. All raw data were subsequently deconvolved using the calculated point spread function (PSF) of the system and the Classic Maximum Likelihood Estimation (CMLE) algorithm with Huygens Professional (SVI,15.10.1). In brief, after an automatic background correction, the signal to noise ratio was set to 15 and the optimized iteration mode of the CMLE was run until a quality threshold of 0.05 was reached. The deconvolved datasets were corrected for a chromatic aberration in z, using the Chromatic Aberration Corrector (CAC) in Huygens.

The co-localization analysis was performed on the deconvolved STED stacks using Imaris 8.3 (Bitplane, Oxford Instruments). To detect punctate staining as spots Imaris spot detection algorithm was applied as follows: the sensitivity for the detection of the spots in each channel was determined by an automatically generated threshold and the spots diameter was set to 0.06 μm. The distances between the spots in the two channels were measured using a customized version of the Imaris XTension Spots Colocalize, which determines the co-localization between the spots within a user-defined distance (1 μm) and bins the data into several bins with equal width (100 nm).

For quantifications, the same detector settings were used for all coverslips quantified in one experiment. From each culture, images from at least two different coverslips were acquired and quantified to minimize experimental variability. The nuclear fluorescence was assessed as established before (Ivanova et al., 2015). ImageJ (NIH) and OpenView software (Tsuriel et al., 2006) were used for quantitative immunofluorescence analysis. After removing the background by threshold subtraction in ImageJ, synaptic puncta were defined with OpenView software by setting rectangular regions of interest (ROI) with identical dimensions around local intensity maxima in the channel with staining for synapsin or any of the other synaptic markers that were used (GluA, homer1, synaptophysin, SV2B). Mean immunofluorescence (IF) intensities were measured in the synaptic ROIs in all corresponding channels using the same software and normalized to the mean IF intensities of the control group for each of the experiments. The number of synapses per unit of dendrite length was determined as follows: First synapsin puncta along 30 μm of proximal dendrite, was detected using Find Maxima function in ImageJ, by setting the same noise tolerance to all images quantified in one experiment; Mean IF intensities of GluA were measured in circular ROIs set around the local intensity maxima in the image with synapsin staining; The number of GluA puncta co-localizing with synapsin was calculated by applying an identical intensity threshold for GluA detection between the different conditions within an experiment.

The pHluorin imaging was performed with hippocampal cultures DIV16 to 20, transduced with lentiviral particles on the day of plating.

The coverslips were removed from the cell culture plates and mounted in an imaging chamber (Warner instruments), supplied with a pair of platinum wire electrodes, 1 cm apart, for electrical stimulation. The imaging was performed at 26°C in extracellular solution, containing 119 mM NaCl, 2.5 mM KCl, 25 mM HEPES pH7.4, 30 mM glucose, 2 mM MgCl2 and 2 mM CaCl2, 10 μM 6-cyano-7‐nitroquinoxaline-2,3-dione disodium (CNQX, Tocris) and 50 μM d-(-)-2‐amino-5‐phosphonopentanoic acid (APV, Tocris), on inverted microscope (Observer. D1; Zeiss-as described above) equipped with an EMCCD camera (Evolve 512; Photometrics) controlled by MetaMorph Imaging (MDS Analytical Technologies) and VisiView (Visitron Systems GmbH) software, using 63x objective. EGFP ET filter set (exciter 470/40, emitter 525/50, dichroic 495 LP, Chroma Technology Corp.) and Cy5 ET filter set (exciter 620/60, emitter 700/75, dichroic 660 LP, Chroma Technology Corp.) were used for imaging of the pHluorin and CypHer5E, respectively. Cultures were stimulated with a train of 40 or 200 action potentials (1 ms, constant voltage pulses) at 5, 20 or 40 Hz using S48 stimulator (GRASS Technologies). The alkaline trapping method was used for quantification of the recycling vesicle pools. In brief, the stimulation of sypHy expressing neurons was done in presence of bafilomycin A1 (1 μM, Merck/Millipore), a specific inhibitor of the vesicular V-type ATPase. Exocytosis of RRP was triggered by delivering of 40 AP at 20 Hz. Following a 2 min break after the end of the first train of stimuli TRP was released by stimulation with 200 AP at 20 Hz. The relative sizes of RRP and TRP were determined as fractions of the total sypHy-expressing pool measured after addition of alkaline imaging buffer (60 mM NaCl in the extracellular solution was replaced with 60 mM NH₄Cl). Fluorescent images were acquired at 1 Hz (Figure 1I) and 10 Hz (Figures 1F, 1J, 1K, ​1K,4E,4E, ​E,6A–6D,6A–6D, S2C, S2G, and S4). Imaging of hippocampal neurons transfected with syp mOrange2 (Figure 4C) was performed in a modified extracellular solution (136-mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1.3 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, 10 μM CNQX, 50 μM APV, pH 7.4) on inverted Zeiss Axio Observer.Z1 epifluorescence microscope, equipped with Zeiss AxioCam 506 camera controlled by ZEISS ZEN 2 software, using EC Plan-Neofluar 40x oil immersion objective (NA 1.3) and a DsRED filter set (exciter 538-562, beam splitter 570, emitter 570-640). Cultures were stimulated with a train of 200 AP delivered at 20 Hz (100 mA, 1 ms pulse width) and fluorescent images were acquired at 0.5 Hz. Synaptic puncta responding to stimulation were identified by subtracting an average of the first several frames of the baseline from an average of several frames at the end of stimulation. The mean IF intensities were measured in ROIs with an identical size, placed automatically over each responding synapse using a self-written macro in ImageJ. The data traces were determined after removing the background by threshold subtraction and correction for bleaching, calculated from the bleaching of unresponsive boutons from the same coverslip. The half times for endocytosis (t1/2) were determined by applying a single exponential fit to the decay phases of the data traces using GraphPad Prism5 and the following equation: Ft = Fstim^∗exp(-t/tau), t1/2 = ln(2)^∗tau, where Fstim is the fluorescence intensity at the end of stimulation and tau is the time constant for endocytosis.

Whole-cell voltage clamp recordings were performed between 14 and 18 days in vitro (DIV) in autaptic neurons at room temperature. Ionic currents were acquired using a Digidata 1440A digitizer and a Multiclamp 700B amplifier under the control of Clampex X software (Axon instrument). Series resistance was set at 70% and only neurons with series resistances below 10 MΩ were selected. Data were recorded at 10 kHz and low-pass filtered at 3 kHz. Borosilicate glass pipettes with a resistance around 3 MΩ were used and filled with an intracellular solution containing (in mM): 136 KCl, 17.8 HEPES, 1 EGTA, 4.6 MgCl₂, 4 Na₂ATP, 0.3 Na₂GTP, 12 phosphocreatine, and 50 U/ml phosphocreatine kinase; 300 mOsm; pH 7.4. Autaptic neurons were continuously perfused with standard extracellular solution composed of (in mM): 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 2 CaCl₂, 4 MgCl₂; 300 mOsm; pH 7.4. Spontaneous release was measured by recording mEPSC for 30 s at a holding potential of −70 mV in the presence of 3 mM kynurenic acid to detect false positive events and for the equal amount of time in extracellular solution. Data were filtered at 1 kHz and analyzed using template-based miniature event detection algorithms implemented in the AxoGraph X software. Action potential-evoked release EPSCs were elicited by 2 ms somatic depolarization from −70 to 0 mV. To estimate the readily-releasable pool (RRP) size, 500 mM hypertonic sucrose added to standard extracellular solution, was applied for 5 s using a fast-flow system (Pyott and Rosenmund, 2002). For vesicular release probability (Pvr) calculations, the ratio of EPSC charge to RRP charge was determined. Short-term plasticity was examined either by evoking 2 unclamped AP with 25 ms interval (40 Hz) or a train of 50 AP at an interval of 100 ms (10 Hz). All electrophysiological data were analyzed offline using Axograph X (Axograph Scientific).