Hippocampal neuron cultures were prepared from E18 rat embryos using previously described methods (Goslin et al., 1998
). Neurons were grown on coverslips inverted over an astroglia feeder layer in serum-free media. Neurons were treated with either 100 μM APV (Research Biochemicals) or 10 μM MK-801 (Alexis) beginning on day 7. Neurons were transfected at 9 DIV using Lipofectamine 2000 (Invitrogen).
COS-7, CV1, and HEK cell lines were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. For fibroblast-neuron cocultures, COS-7 or CV1 cells were transfected with Effectene (Qiagen) and trypsinized 24 hr later. Cells were washed twice, plated onto neurons, and the cocultures were maintained for 24 hr before fixation.
Soluble neurexin-Fc (human IgG) or neurexinΔLNS-Fc were expressed in COS cells. Protein was collected in serum-free media, purified with protein A columns from an ImmunoPure IgG purification kit (Pierce), and concentrated in PBS with Centricon filters (Millipore). For bead experiments, nonfluorescent Neutravidin labeled FluoSpheres (Molecular Probes) with a diameter of 1 μm were incubated with either biotin conjugated anti-GFP (here called anti-YFP; Rockland Immunochemicals) or biotin conjugated anti-human IgG (Jackson ImmunoResearch). The beads were rinsed in PBS, and the anti-human IgG bound beads were further incubated in either soluble neurexin-Fc or soluble neurexinΔLNS-Fc. One day after beads had attached to the neurons, the cells were fixed and stained.
The expression vector for soluble neurexin-1β-Fc containing aa 1–262 of mouse neurexin-1β was received from P. Scheiffele (Columbia University; Scheiffele et al., 2000
). Soluble neurexinΔLNS-Fc was created by deleting the entire LNS domain (aa 84–261). To create neurexin-1β-CFP, aa 262–438 of neurexin-1β were cloned by PCR from Marathon rat brain cDNA (Clontech). The resulting product was fused to aa 1–262 from neurexin-1β-Fc and inserted into pECFP-N1 (Clontech) to create a full-length neurexin-1β construct with CFP attached to the C terminus. This form of neurexin-1β lacks any insert at splice site 4. The β-neurexin-specific region was deleted by removing aa 47–83. To delete the LNS domain, aa 84–261 were excised. The entire glycosylated region was deleted by removing aa 262–356. Removal of either aa 262–311 or 312–356 created partial deletions of the glycosylated region. To replace the neurexin LNS domain with the agrin LNS domain, aa 84-261 of neurexin-1β were replaced with aa 1863-2043 of human agrin (XM_372195; cDNA provided by J. Sanes, Harvard University). To determine synaptic localization of CFP-neurexin in neurons, the CFP tag was removed from the C terminus and inserted into the extracellular β-neurexin-specific region between residues 63 and 64. Expression vector for full-length agrin from rat was a gift from M. Ferns (University of California, Davis), for N-cadherin-YFP was a gift from D. Benson (Mount Sinai School of Medicine), and chick NgCAM was a gift from Peter Sonderegger (University of Zurich). NgCAM-YFP was made by attaching YFP to the C-terminal end of the protein. The HA-tagged neuroligins-1 and -2 (gift from P. Scheiffele; Scheiffele et al., 2000
) both contain the neuroligin-1 signal sequence followed by the HA tag (YPYDVPDYA) and the mature N terminus of each of the neuroligins inserted into the pNice expression vector. YFP-neuroligin-1 and -2 were created by removing the HA epitope in HA-neuroligin-1 and -2 and inserting YFP in between the signal peptide and the mature N terminus. YFP-neuroligin-3 and -4 were created by replacing the neuroligin-2 sequence following YFP with the mature neuroligin-3 and -4 sequences from human neuroligin-3 (BC051715, Open Biosystems) and human neuroligin-4 (BC034018, Open Biosystems). Rat PSD-95 (gift from M. Sheng, MIT) was fused at its C terminus with CFP and expressed from the Clontech pECFP-N1 vector. The HA-CD8 construct contains the neuroligin-1 signal sequence followed by the HA-tag (YPYDVPDYA) and the entire mature human CD8α sequence (NM_001768).
The following mouse monoclonal antibodies were used: gephyrin (mAb7a; IgG1; 1:500; Alexis), PSD-95 (although the term PSD-95 is used for simplicity, this antibody recognizes PSD-95, PSD-93, SAP102, and SAP97; 6G6-1C9; IgG2a; 1:500; Affinity Bioreagents), NR1 (54.1; IgG2a; 1:1000; PharMingen), α-dystroglycan (VIA4.1; IgG1; 1:50; Upstate Biotechnology), β-dystroglycan (43DAG1/8D5; IgG2a; 1:25; Novocastra), GAD65 (GAD6; IgG2a; 1:100; Developmental Studies Hybridoma Bank), MAP2 (IgG1; 1:500; Chemicon), HA (IgG2b; 1:500; Roche). Rabbit antibodies were used against synapsin (1:1000; Chemicon), MAP2 (1:20,000; gift from S. Halpain, Scripps Research Institute), the γ2 subunit of GABAA receptors (1:200; Alomone), GluR1 (1:5000; Upstate Biotechnology), SynGAP (1:1000; Affinity Bioreagents), and GKAP (1:300; gift from M. Sheng, MIT). Guinea pig antibodies were used against the α2 and γ2 subunits of GABAA receptors (1:2000; gift from J.M. Fritschy, University of Zurich) and VGlut 1 (1:2000; Chemicon). Goat antibodies were used against neuroligin-2 (D-15; 1:200; Santa Cruz Biotechnology) and agrin (1:1000; R&D Systems). The specificity of this neuroligin-2 antibody was confirmed by immunoreactivity against COS cells expressing YFP-neuroligin-2 but not YFP-neuroligin-1 (data not shown). Secondary antibodies were Alexa488, Alexa568, and Alexa647 conjugated anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-rabbit, anti-guinea pig, and anti-goat (Molecular Probes), and AMCA anti-rabbit (Vector Laboratories).
Generally, neurons were fixed for 15 min in warm PBS with 4% paraformaldehyde and 4% sucrose and permeabilized with 0.25% Triton X-100. They were incubated in 10% BSA (30 min, 37°C), appropriate primary antibody (Ab) in PBS with 3% BSA (overnight, 20°C) and secondary Ab (45 min, 37°C). Coverslips were mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol with 2% 1,4-diazabicyclo[2,2,2]octane). Coverslips used for NR1, β-dystroglycan, or neuroligin-2 immunostaining were fixed with methanol for 10 min at −20°C. When used with other antibodies, neuroligin-2 immunostaining was performed sequentially, with neuroligin-2 antibody first. Fluorescence and phase-contrast images were captured with a Photometrics Sensys or Diagnostics Instruments SPOT-RT cooled CCD camera mounted on a Zeiss Axioscope or Axioplan microscope with a 63× 1.4 NA oil objective using Metamorph imaging software. To determine surface localization of mutated neurexin-CFP proteins in transfected HEK cells and colocalization of induced neuroligin-2 with gephyrin and PSD-95, optical sections were captured using an Olympus Fluoview FV500 confocal on a BX61W microscope with a 60× 1.4 NA oil objective. Imaging in sequential scan mode with 442, 488, 568, and 633 nm laser lines and customized filters was used for separate detection of fluorophores. Images were prepared using Adobe Photoshop 5.5 software.
Neurons were transfected at 9 DIV to express HA-neuroligin-2 or HA-CD8 along with YFP. Neurons were identified for recording by expression of YFP and without knowledge of the cotransfected protein. Spontaneous mPSCs were recorded at 12–14 DIV at room temperature in the whole-cell voltage-clamp configuration. The extracellular solution contained: 168 mM NaCl, 2.4 mM KCl, 10 mM HEPES, 10 mM D-glucose, 1.3 mM CaCl2, and 1.3 mM MgCl2 (pH 7.4). mEPSCs were recorded in the presence of 0.5 μM TTX, 100 μM APV, and 10 μM SR95531. For mEPSCs, the intracellular solution contained: 140 mM Kgluconate, 10 mM HEPES, 8 mM NaCl, 2 mM Na2ATP, 0.1 mM Na2GTP, 10 mM EGTA, 6.23 mM CaCl2, and 2 mM MgCl2 (pH 7.4). mIPSCs were recorded in the presence of 0.5 μM TTX, 100 μM APV, and 5 μM NBQX. For mIPSCs, the intracellular solution contained: 144 mM CsCl2, 10 mM HEPES, 5 mM Na2ATP, 1.1 mM EGTA, 0.1 mM CaCl2, and 5 mM MgCl2 (pH 7.4). Recordings were performed with an Axopatch-1D amplifier and Axograph software (Axon Instruments). Records were filtered at 5 kHz and acquired at 10 kHz. Neurons were clamped at −70 mV. Series resistance values were <10 MΩ, input resistance >150 MΩ, and these parameters did not differ between groups. mPSCs were detected with Axograph software event detection using an optimally scaled sliding template and criteria of three times the SD.
Sets of cells used for quantification were stained simultaneously. Images were randomized prior to quantification so the experimenter was blind to the treatment group. For the neuron-fibroblast coculture assay, transfected COS cells were chosen randomly based on phase contrast showing significant contact of COS cells with dendrites. Images were taken of the postsynaptic proteins, the presynaptic protein, and the transfected COS cell (neurexin-CFP or mCFP) using the same exposure time for both experimental and control conditions. Images of the pre- and postsynaptic proteins were thresholded and the area for measuring defined by the perimeter of the transfected COS cell. For each postsynaptic protein cluster, the area and total gray value was measured. A region was drawn around each cluster and thresholded synapsin or VGlut was measured through these regions to determine which clusters were synaptic. Only values from postsynaptic protein clusters that were not apposed to synapsin or VGlut were used in the final quantification.
For assessing colocalization of induced clusters of neuroligin-2 with gephyrin and PSD-95, neurexin-CFP transfected COS cells with induced clusters of gephryin and PSD-95 were selected. Gephyrin and PSD-95 images were randomized and thresholded, and regions were drawn around each cluster. Images of neuroligin-2 were thresholded, and the percentage of gephyrin and PSD-95 puncta that exhibited any pixel overlap with thresholded neuroligin-2 was quantified.
To determine the amount of gephyrin and PSD-95 localized to regions of clustered YFP-neuroligin-1 or -2 by anti-YFP bound beads, transfected neurons exhibiting bead-induced clusters of YFP-neuroligin-1 or -2 were selected by scanning in phase contrast and the YFP channel. YFP-neuroligin images were thresholded to define regions of neuroligin clustered by beads. The average gray value of unthresholded gephyrin and PSD-95 was measured within these regions, and the average gray value of the off-cell background subtracted. These values were then normalized to mean dendrite gephyrin and PSD-95 average gray value, also subtracted for off-cell background, so that final values reflect relative concentrations of gephyrin or PSD-95 above a mean dendrite value of 1.
For quantification of postsynaptic proteins in neurons transfected with HA-neuroligin-2, neurons overexpressing HA-neuroligin-2 or HA-CD8 were chosen randomly based on health and level of expression. Images were thresholded and postsynaptic protein puncta were delineated by the perimeter of the transfected neuron. Random regions of dendrite were selected and the number, area, and total gray value of the thresholded postsynaptic protein puncta per 100 μm length of dendrite were measured.
Analysis was performed using Metamorph, Microsoft Excel, Stat-View, and Cricket-Graph. Statistical comparisons of immunofluorescence were made using Student’s unpaired t test. All data are reported as mean ± SEM.