The design and generation of amiRNAs targeting SynCAM1–3 and Scrambled1–3 amiRNAs, a MultiSite Gateway (Invitrogen) middle entry vector with an intronically-expressed enhanced synthetic inhibitory BIC/miR-155 RNA cloning cassette (pME-eSIBR), and insertion and chaining of amiRNAs in the cassette were described previously (Fowler et al., 2016b). SynCAM1–3 (cadm1–3) guide strand amiRNA targeting sequences used were cadm1.1358:5′-UUGAUUAUAGCUGUGUCUGCGU-3′, cadm2.87:5′-UUCAACAACCGUGACAUUCUGA-3′, and cadm3.387:5′-AUAACCAGUGAUUAUGGGUUUC-3′. Scrambled control guide strand sequences used were scrambled1: 5′-AUUCUAAUACUACGUUCCGCAU-3′, scrambled2: 5′-ACAACUUGUAUAUCGCGCAACU-3′ and scrambled3: 5′-GAUCUUAUACUCGUGAUUGAGA-3′. Gateway LR recombination reactions of pME-eSIBR vectors with single, double, or triple amiRNAs with a 5′ entry vector containing a minimal CMV (mCMV) promoter (p5E-CMVmin) and 3′ entry vector with a nlsGFP tag (p3E-nlsGFP no-pA) into a third-generation lentiviral destination vector (pEpic_Lite) were performed to create SynCAM single, double and triple KD vectors or the control Scrambled1–3 amiRNA vector were described previously (Fowler et al., 2016a). To make the memGFP-only expressing vector, a middle entry vector containing GFP with a C-terminal human H-RAS palmitoylation signal for membrane targeting (Kwan et al., 2007) was used in a MultiSite Gateway LR reaction with p5E-CMVmin, a 3′ entry vector with a hemagglutinin epitope tag (p3E-HA no-pA), and pEpic_Lite (Fowler et al., 2016a). The HA epitope is not expressed because the memGFP sequence used contains a stop codon and was used as a “filler” sequence to allow LR recombination.

2.5 × 10⁶ HEK293T cells (ATCC^® CRL-3216) were plated per 10-cm tissue culture dishes in 10 ml of DMEM (Invitrogen), 10%FCS (Atlanta Biologicals), 25 units/ml penicillin and 25 μg/ml streptomycin (Sigma). Approximately twenty-four hours after plating, cells were transiently transfected using ProFection (Promega) calcium phosphate transfection reagents with 20 μg pEpic_Lite lentiviral vectors and packaging vectors (10 μg pMDL g/p RRE, 5 μg pRSV-Rev, 6 μg pVSV-G; Dull et al., 1998). Six to Eight hours later, media was replaced with 6 ml/plate of fresh medium. Medium was collected 48–72 h after transfection and centrifuged at 3000× g for 5 min at room temperature (RT). Supernatant was passed through a 0.45 μm syringe filter and virus was concentrated by centrifugation on a 150,000 MWCO column (Pierce). Twenty thousand HEK293T cells were plated per well of a 12-well plate and transduced with serial dilutions of concentrated lentivirus. Four to five days after transduction, titers were calculated by flow cytometry on an Attune^® acoustic focusing cytometer (Applied Biosystems) for GFP+ cells. Infectious lentiviral particles/μl was calculated from viral dilutions where cells were transduced in the linear range (5%–20% GFP+ cells).

Studies using rats were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the University of Oregon and Washington State University Institutional Animal Care and Use Committee (Permit Numbers: #13-19 and #04787, respectively). Rats were anesthetized with isoflurane prior to sacrifice and culturing of neurons. Rats were housed with a 12/12 light/dark cycle according to standard protocols in the University of Oregon Animal Care Facility and Washington State University Veterinary and Biomedical Research Vivarium. Sprague-Dawley rats were obtained from Envigo.

Hippocampal cultures were prepared from embryonic day 19 Sprague-Dawley rat pups as described (Brewer et al., 1993), with minimal modifications. For single-cell SynCAM immunofluorescence comparisons, 3000 dissociated hippocampal cells were plated per well of a 12-well plate. For all other experiments, cells were cultured at a density of 100,000 cells/well of a 12-well plate. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments cells were attached directly to plates coated with poly-L-lysine (Sigma); for all other experiments cells were cultured on glass coverslides coated with poly-L-lysine. Cells were allowed to attach to poly-L-lysine coated substrate in plating media (MEM (Invitrogen), 10% FCS, 20 mM dextrose, 25 units/ml penicillin and 25 μg/ml streptomycin) for 5–6 h. Media was then changed to maintenance media (Neurobasal medium (Invitrogen), 1× B-27 supplement (Invitrogen), 0.5 mM Glutamax (Invitrogen), 50 units/ml penicillin, 50 μg/ml streptomycin, and 0.07% β-mercaptoethanol (Sigma)). Half changes of maintenance media were performed every 3–4 days in culture. Primary hippocampal cultures were infected with lentivirus at 1–2 days in vitro (DIV). For saturating transduction with lentivirus, 20,000 infectious lentiviral particles (as calculated by our titration method) were added per well of a 12-well plate; for sub-saturating transduction 2000 infectious lentiviral particles were added. For studies using memGFP lentivirus, 100 infectious particles were additionally added. Cells were fixed and stained at 13–15 DIV for imaging experiments; electrophysiology recordings were made with cells at 13–16 DIV.

Standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) western blotting procedures using nitrocellulose membranes were performed as previously described (Fowler et al., 2016b). Two-color near-infrared blots were imaged with an Odyssey-Fc quantitative western blot system (LI-COR). Primary antibodies and dilutions used were mouse anti-actin 1:2000 (Millipore, clone C4) and rabbit anti-SynCAM1–3 1:1000 (Pierce, PA3-16744); secondary antibodies donkey anti-mouse IRDye 680RD and anti-rabbit IRDye 800CW (LI-COR) were used at 1:1000. Intensities were normalized to actin loading controls. KD efficiency was calculated by comparing levels relative to the Scrambled 1–3 amiRNA control conditions set to 1. The representative blot shown is a composite image made by re-arranging lanes of a single blot image at the same projection intensity.

First-strand cDNAs were synthesized from total RNA isolated from cultured hippocampal neurons using Superscript III reverse transcriptase (Invitrogen) with oligodT primers for 50 min at 50°C. Primer pairs used to measure cadm mRNA levels were cadm1_F: 5′-GAAGGACAGCAGGTTTCAGC-3′, cadm1_R: 5′-ACCAGGACTGTGATGGTGGT-3′, cadm2_F: 5′-TCCTGATCGAATGGTTGTGA-3′, cadm2_R: 5′-TGGGATCGTGTACAATGAGG-3′, cadm3_f: 5′-CCTGGAGAAAAGGTGACCAA-3′, cadm3_R: 5′-ATGGTTCACAGAGCACACGA-3′. qRT-PCR was performed using SYBR Green reagents (Kapa Biosystems) using standard parameters on a StepOnePlus Real-Time PCR System (Applied Biosystems). Values and relative expression levels were compared using the ΔΔC_t method.

Cells on glass coverslides were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 15 min at 4°C. Cells were then permeabilized for 5 min with 0.25% Triton-X100 in PBS, and blocked for 1 h at RT with blocking solution (1% Roche blocking solution (Roche), 10% BSA (Sigma), 1% normal donkey serum (Jackson ImmunoResearch), and 1% normal goat serum (Jackson ImmunoResearch) in PBS). Cells were then incubated with primary antibodies in blocking solution overnight at 4°C. Primary antibodies and dilutions used were rabbit anti-Synapsin1 1:500 (EMD Millipore, AB1543), mouse anti-PSD-95 1:350 (Neuromab, clone K28/43), chicken anti-GFP 1:2000 (Aves Labs, GFP-1020), and rabbit anti-SynCAM1–3 1:500 (Pierce, PA3-16744). The next day, cells were washed 3× 5 min with PBS and incubated with secondary antibody in blocking solution for 1 h at RT. All secondary antibodies were from Jackson Laboratories and used at 1:500–goat anti-chicken Alexa Fluor 488, donkey anti-mouse or anti-rabbit Cy3, and donkey anti-rabbit Cy5. Cells were washed 3× 5 min with PBS and mounted on slides with Fluoromount G with DAPI (Southern Biotech).

For transduction rate experiments, neurons were imaged on a Nikon Eclipse TE300 microscope using a 20× air objective (0.45 NA), Till Photonics monochromator light source, Retiga EXi CCD camera (Q Imaging) and SimplePCI software (Hamamatsu, Inc., Houston, TX, USA). For Figures 3B,C, cells were imaged at 20× (Figure 3B) using an air (0.75 NA) objective or at 60× (Figure 3C) using an oil-immersion objective (1.40 NA) on an Eclipse 80i microscope with a DS-Qi1Mc camera, Intensilight C-HGFI light source and Elements software (Nikon). For Figure 3D, cells were imaged live in aCSF (see “Electrophysiology” Section for recipe) with a 40× water-immersion objective (0.8 NA) using an Eclipse FN1 microscope with a DS-Qi1Mc camera, Intensilight C-HGFI light source and Elements software (Nikon). All other neurons were imaged on an inverted Nikon TU-2000 confocal microscope using EZ-C1 software. For single-cell comparisons of SynCAM1–3 immunofluorescence, images were obtained using a 20× air objective (0.75 NA) and the example images in Figures 4A,B were obtained using a 60× water-immersion objective (1.2 NA). All other images were obtained with a 100× oil-immersion objective (1.45 NA). The presence or absence of nlsGFP was validated by visual comparison of DAPI and GFP staining. For transduction rate comparisons, 20 images of DAPI (350 nm) and corresponding GFP (488 nm) signal were taken at random positions across four coverslides per condition. For single-cell SynCAM1–3 immunofluorescence experiments up to 20 neurons that did not overlap with neighboring cells were selected for imaging for each condition. Eight neurons were imaged for a secondary antibody-only condition for analysis of background fluorescence. Sequential scanning for each channel (488, 543 nm) was performed and the average of three scans was taken at 1024 × 1024 pixel resolution. For all other experiments, pyramidal cells were selected by morphology and cells were imaged if they had 2–3 primary or secondary small diameter dendrites (~1–2 μm not including spines or protrusions) that originated at the soma with no further branches in a single field of view at high magnification, that terminated within a short distance of the soma (usually <100 μm), and that were visually discernable from additional GFP-positive processes and background immunofluorescence. Cells were sampled evenly between isolations. Sequential scanning for each channel (488, 543, 633 nm) was performed and the average of three scans was taken at 2048 × 2048 pixel resolution. For all experiments, images were obtained with constant pinhole, laser intensity, and detector gain settings, and the experimenter was blinded to the conditions.

Each color channel was saved independently as grayscale 16-bit TIFF files. For transduction rate comparisons, the number of total cells in an image were determined by manually counting DAPI nuclei; the number of transduced cells was determined by manually counting nlsGFP+ nuclei. For single-cell SynCAM1–3 KD comparisons, binary masks were made of each neuron using Image-Pro 6.3 software (Media Cybernetics) using outlines from 488 nm images. Background debris was cleared from the masks manually using GIMP 2 software (The GIMP Team). SynCAM immunostaining intensity was calculated within the confines of the outline of the neuron defined by the binary masks using a custom program in MATLAB (Mathworks). The average fluorescence intensity for neurons in the secondary antibody only condition was used to measure background signal and was subtracted from the SynCAM staining intensity for each image. For dendrite analysis, using 488 nm images, individual basal dendrite segments averaging ~30 μm in length were selected and binarized manually using Image-Pro 6.3 software (Figures 2A,B). Binary masks were then used in a custom MATLAB program to automatically detect and compare puncta from corresponding 543 nm and 633 nm images (Figure 2B). Briefly, the program calculated the average fluorescent intensity of corresponding images in the binarized GFP region, set a threshold for including pixels in puncta detection (1.5× the mean value for each dendrite), and automatically detected puncta that were >4 contiguous pixels within the confines of the binarized GFP mask. Only puncta >0.15 μm² were considered for density and size analysis. Overlapping pre- and postsynaptic puncta were counted as synapses. Dendrite lengths were measured manually using Image-Pro 6.3 software. Puncta density was calculated by dividing number of puncta by dendrite length. Puncta area was reported by our custom program. Experimenters were blinded to conditions during image analysis.

Whole cell recordings were performed on identified pyramidal neurons in hippocampal cultures using an upright Nikon FN1 microscope with fluorescence imaging capabilities. Recording electrodes (2.8–3.8 MΩ) were filled with an intracellular solution containing (mM): 6 NaCl, 4 NaOH, 130 Cs-gluconate, 11 EGTA, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 2 Na₂ATP, and 0.2 Na₂GTP. The intracellular solution was pH 7.4 and 296 mOsm. All neurons were studied under voltage clamp conditions with an Axopatch 200A or MultiClamp 700A amplifier (Molecular Devices). Neurons were held at V_H = −70 mV using pipettes in whole cell patch configuration. Signals were filtered at 3 kHz and sampled at 30 kHz using p-Clamp software (version 10, Molecular Devices). Liquid junction potentials were not corrected. Extracellular solution (aCSF; containing (mM): 125 NaCl, 3 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 10 dextrose, and 2 CaCl₂) was continuously perfused and drugs were bath applied to isolate quantal glutamatergic signaling (TTX, 1 μM and Gabazine, 3 μM).

Digitized waveforms of quantal synaptic events were analyzed using MiniAnalysis software (Synaptosoft). Only traces from cells held under voltage clamp with a series resistance <25 MΩ were used for analysis. All events >5 pA were counted for 1–2 min of trace ~5 min after application of drug. All events were used to calculate frequency values. The average mEPSC projection of all discrete events for each neuron was used for peak analysis measurements by automated fitting of amplitudes and decay kinetics (single exponential, 90–10%) with MiniAnalysis software. Noisy, misaligned and non-discrete events were manually removed prior to peak analysis. The experimenters were blinded to the conditions for recording and trace analysis.

Normality of data was determined by Shapiro-Wilk tests in R (R Foundation for Statistical Computing). P values obtained by statistical comparisons of two sample groups used Student’s two-tailed, unpaired t-tests in Microsoft Excel and comparisons of more than two sample groups used one-way ANOVAs followed by Tukey’s post hoc pairwise comparisons in R.

Single and multi-gene KD was achieved through the use of inhibitory RNA (RNAi) targeting sequences in an enhanced amiRNA backbone either singly or chained in a mobile cassette (Fowler et al., 2016b). We combined amiRNAs targeting rat SynCAM1–3 in single, double and triple-gene KD combinations, as well as three scrambled amiRNA sequences targeting no known genes (Scrambled1–3), into a lentiviral destination vector with a nuclear localized GFP (nlsGFP) reporter (Fowler et al., 2016a; Figure 1A). These amiRNAs were expressed from an intron to prevent degradation of the mRNA to allow robust reporter expression (Chung et al., 2006). We also generated a separate lentiviral vector driving expression of a plasma membrane-localized GFP (memGFP) reporter used to label dendrites and spines (Figure 1B). We previously characterized these chained amiRNAs targeting SynCAM1–3, which are encoded by the cell adhesion molecule 1–3 (cadm1–3) genes, as highly efficient for KD of SynCAM1–3 in rat primary hippocampal cultures at 13–15 DIV by western blot (Fowler et al., 2016b). For further validation, we performed qRT-PCR for cadm1–3 mRNA levels in hippocampal cultures following transduction with saturating levels of SynCAM1–3 amiRNAs. Results showed >90% KD of cadm1 and 3, and >80% KD of cadm2 mRNAs, confirming that our amiRNAs are effective for KD of all three SynCAMs (Figure 1C).

Typically, Lentiviral RNAi experiments are performed at saturating transduction levels to ensure the highest level of KD. However, saturating amiRNA virus prevents a population of non-transduced cells retaining WT SynCAM1–3 expression levels. Because we eventually wanted to compare synapse development in both WT and SynCAM1–3 KD neurons on the same coverslip, we infected cultures with sub-saturating amounts of SynCAM1–3 amiRNA or control Scrambled1–3 amiRNA virus. We counted nlsGFP+ DAPI-stained nuclei and saw that ~80% of cells were transduced in both cases (Figure 1D), ensuring that ~20% of cells retained WT SynCAM expression levels.

Because our previous characterizations of SynCAM1–3 KD by western blot (Fowler et al., 2016b) and in the current study with qPCR (Figure 1C) used saturating viral levels, we wanted to confirm that sub-saturating transduction still produced robust KD. Using quantitative western blotting with an antibody that recognizes SynCAM1–3 (Biederer et al., 2002), but not SynCAM4 (Fogel et al., 2007), we showed ~90% KD with saturating and ~70% KD with sub-saturating virus (Figures 1E,F). Because around 20% of cells retained WT SynCAM1–3 expression in sub-saturating virus, we reasoned that the observed 70% KD likely underrepresented the actual KD amount in SynCAM1–3 amiRNA+ cells. Therefore, we cultured cells at very low densities to allow imaging of individual cells transduced with sub-saturating concentrations of SynCAM1–3 or Scrambled1–3 amiRNA lentivirus. As measured by immunofluorescence intensity for SynCAM1–3 antibody, SynCAM1–3 amiRNA+ cells had ~95% KD compared to Scrambled1–3 amiRNA+ cells (Figures 1G,H). This confirmed that sub-saturating concentrations of the amiRNA lentivirus effectively eliminated targeted protein expression in individual transduced neurons, while maintaining a subpopulation of amiRNA− cells to be used as in-culture WT controls.

To test functional redundancy of SynCAM1–3 during synapse formation, we infected hippocampal cultures at sub-saturating amounts with all possible combinations of lentivirus carrying single, double, and triple amiRNAs against SynCAM1–3, as well as the Scrambled1–3 amiRNA control. We also infected a small subset of neurons (~1%) with memGFP lentivirus to enable imaging of dendrites. To detect differences in synapse formation, we immunolabeled cultures at 13–15 DIV with antibodies to GFP, the postsynaptic protein PSD-95 and the presynaptic protein Synapsin 1 and imaged nlsGFP/amiRNA+ cells that were also labeled with memGFP using confocal microscopy (Figure 2A). Following manual selection of dendrites on pyramidal cells, we used automated detection of PSD-95 and Synapsin 1 puncta. We defined synapses as regions of co-localization of these pre- and postsynaptic puncta along dendrites (Figure 2B). Only SynCAM1–3 KD, but not single or double KD, reduced synapse density by nearly 20% (Figure 2C) and increased the area of co-localization by ~10% (Figure 2D), demonstrating that SynCAM1–3 function redundantly to set synapse number and size in developing neurons.

We sought to determine if the phenotypes due to SynCAM1–3 KD are caused by pre- or postsynaptic loss. Sub-saturating infection with amiRNA lentivirus results in non-transduced cells retaining WT SynCAM1–3 expression levels. Combinatorial application of memGFP lentivirus enables labeling of these WT cells for imaging, which is not possible when an RNAi payload is directly linked to the neurite-labeling fluorophore. Therefore this new methodology, named Mosaic Expression using Differentially Localized Reporters (MEDLR), allows direct comparisons of WT (memGFP+ only) and KD (memGFP+/nlsGFP+) neurons on the same coverslip (Figure 3A). Using MEDLR, amiRNA− WT cells were readily distinguished from amiRNA+ KD cells when visually scanned for the presence or absence of nlsGFP fluorescence in memGFP-labeled fixed cell preparations under low magnification (Figure 3B). The distinction between amiRNA− and amiRNA+ cells was even more apparent at higher magnifications (Figure 3C). Further, MEDLR was used to differentiate amiRNA− and amiRNA+ cells in live cultures (Figure 3D), showing that this methodology is suitable for approaches such as live cell imaging and electrophysiology.

Crucially, for proteins located on both axons and dendrites, such as SynCAM1–3, MEDLR allows the discrimination between pre- and postsynaptic sites of action. This is because in a culture infected with sub-saturating SynCAM1–3 amiRNAs, the majority of axons (~80%) available to form connections with WT neurons have ~5% of wildtype levels of SynCAM1–3. Therefore if presynaptic SynCAM1–3 is necessary for proper synapse formation then WT cells would be expected to display a similar phenotype to SynCAM1–3 KD neurons. However, if postsynaptic SynCAM1–3 instructs synapse development, no phenotype would be expected in WT cells. We note that as used here, MEDLR cannot determine if postsynaptic SynCAM1–3 is necessary for synapse formation, because in amiRNA+ cells both pre- and postsynaptic SynCAM1–3 would be removed. Instead, MEDLR can determine if the presence of postsynaptic SynCAM1–3 is sufficient for synapse formation.

We used MEDLR to compare synaptic phenotypes between amiRNA− and amiRNA+ cells in cultures transduced with sub-saturating amounts of Scrambled1–3 or SynCAM1–3 amiRNA lentivirus (Figure 4A). WT amiRNA− cells did not display reduced synaptic density compared to Scrambled1–3 amiRNA+ neurons (Figures 4B,C) suggesting that the reduction in SynCAM1–3 amiRNA+ neurons was due to depletion of postsynaptic SynCAM1–3. Further, analysis of PSD-95 (Figures 4D,E) and Synapsin 1 puncta density (Figures 4F,G) showed that both were reduced along dendrites of SynCAM1–3 amiRNA+ neurons but not on Scrambled1–3 amiRNA+ neurons or amiRNA− cells, positing that reduced synapse density is due to the inability to recruit both pre- and postsynaptic structures. MEDLR also revealed no difference in synapse area in amiRNA− cells compared to Scrambled1–3 amiRNA+ neurons (Figures 4H,I), again suggesting that the increased synapse size is because of postsynaptic SynCAM1–3 loss. In contrast to puncta density, there was no difference in mean PSD-95 (Figure 4J) or Synapsin 1 (Figure 4L) puncta size between Scrambled1–3 and SynCAM1–3 amiRNA− or amiRNA+ cells, although we note that distribution plots indicated a subset of amiRNA+ cells that had larger mean PSD-95 and Synapsin 1 puncta areas (Figures 4K,M). Given that roughly twice as many Synapsin 1 and PSD-95 structures are detected than synapses, we cannot rule out that subtle increases in the size of Synapsin 1 and PSD-95 puncta size in SynCAM1–3 KD cells may contribute to the observed, enlarged synapse size. Alternatively, the increased synapse area may be due to the increased overlap of a subset of pre- and postsynaptic markers, which would be indicative of an effect primarily at trans-synaptic complexes. Further, it should be noted that increased puncta overlap in SynCAM1–3 amiRNA+ cells is unlikely to be from a non-specific effect that generally brings puncta in closer apposition, because individual puncta density was also decreased in these cells (Figures 4D–G).

The MEDLR approach enabled us to determine the extent to which quantal glutamatergic synaptic transmission was altered in SynCAM1–3 KD neurons. Using whole-cell patch clamp electrophysiology, we measured mEPSCs at 13–16 DIV onto amiRNA− and amiRNA+ neurons transduced with sub-saturating amounts of Scrambled1–3 or SynCAM1–3 amiRNAs (Figure 5A). Distributions of average mEPSC frequencies across cells were highly variable, logarithmically distributed, and spanned nearly two orders of magnitude (Figure 5B). Because of the spread of frequencies, we did not observe a systematic difference between any group suggesting that SynCAM1–3 do not significantly impact the net frequency of quantal release. This result highlights that quantal release frequency is a product of multiple factors, including intrinsic release probability (Branco and Staras, 2009), in addition to synapse density, and is not necessarily an accurate measure of synapse number.

We also compared mEPSC event traces between groups. On average, mEPSCs were larger in SynCAM1–3 KD neurons compared to Scrambled1–3 amiRNA+ cells (Figure 5C). Waveform fitting analysis showed that while peak amplitude was not different between Scrambled1–3 and SynCAM1–3 amiRNA+ cells (Figure 5D; p = 0.36, t test), peaks in SynCAM1–3 KD neurons tended to have a larger area (Figure 5E; p = 0.14, t test) and had significantly longer rise times and decay-time constants (Figures 5F,G); indicating that SynCAM1–3 KD causes broader mEPSCs. MEDLR showed no differences between amiRNA− cells and Scrambled1–3 amiRNA+ neurons, demonstrating that postsynaptic depletion of SynCAM1–3 enlarges mEPSCs (Figures 5D–G). Additionally, there was no difference in average cell capacitance between conditions (Scrambled amiRNA+ = 52.5 ± 3.2 pF, n = 25; Scrambled amiRNA− = 55.3 ± 3.5 pF, n = 14; SynCAM1–3 amiRNA+ = 55.8 ± 3.4 pF, n = 32; SynCAM1–3 amiRNA− = 54.0 ± 4.8 pF, n = 20), indicating that changes in mESPC kinetics was not due to a change in cell size. Collectively, these results are consistent with an increased synapse size caused by postsynaptic loss of SynCAM1–3, and lend further evidence that defining synapses as juxtaposed pre- and postsynaptic puncta accurately represents functional contacts.