Ethics statement

All animal experiments were approved by the institutional ethical committees and carried out in accordance with the guidelines for animal experimentation in RIKEN, Nagasaki University Graduate School of Biomedical Sciences, and Gunma University Graduate School of Medicine.

Antibodies

The following antibodies were purchased from the indicated companies: actin (A2066, Sigma, 1:1,000 dilution for IB), PSD-95 (MA1-045, Thermo Scientific, 1:5,000 dilution for IB; 1:100 dilution for immunocytochemistry), synaptophysin (S5768, Sigma, 1:10,000 dilution for IB; 1:200 for immunocytochemistry), synapsin-I (Millipore, 1:1,000), somatostatin (sc-7819, Santa Cruz, 1:2,500 dilution for immunofluorescence staining of vibratome sections; 1:250 dilution for immunostaining of frozen sections), parvalbumin (P3088, Sigma, 1:5,000 dilution for immunofluorescence staining of vibratome sections; 1:4,000 dilution for immunostaining of frozen sections), gephyrin (mAb7a, Synaptic Systems, 1:250 dilution for immunocytochemistry), GAD67 (MAB5406, Millipore, 1:250 dilution for immunocytochemistry), mGluR7 (07-239, Millipore, 1:2,000 dilution for IB; 1:100 dilution for immunocytochemistry; 1:2,000 dilution for immunofluorescence staining of vibratome sections), mGluR1a (G209-488, BD Pharmingen, 1:1,000 for immunocytochemistry; 1:2,000 dilution for immunofluorescence staining of vibratome sections), GluR5 (N-19, Santa Cruz, 1:1,000 for IB), PICK1 (N-18, Santa Cruz, 1:1,000 for IB), Vglut1 (AB5905, Millipore, 1:400 for immunocytochemistry; 1:500 for immunostaining for frozen section), Myc (9E10, Santa Cruz, 1:500 dilution for IB), HA (12CA5, Roche, 1:500 for IB), Fc (109-035-098, Jackson Immunoresearch, 1:10,000), and GFP (3E6 and chicken polyclonal, Invitrogen, 1:500 dilution for immunocytochemistry). For production of anti-Elfn antibodies, rabbits were immunized with a synthesized C-terminal Elfn1 peptide (aa 817–828), purified Elfn1–GST (glutathione S-transferase) or Elfn2–GST fusion protein by following a standard protocol. cDNA fragments including the C-terminal domains of mouse Elfn1 (aa 635–828) and mouse Elfn2 (aa 545–823) were subcloned into the pGEX-6P2 plasmid (GE Healthcare) for expression of GST fusion proteins in E. coli BL21 (DE3). The GST fusion proteins were purified using glutathione Sepharose 4B (GE Healthcare) and used for immunization. The obtained antisera were purified by affinity chromatography with the peptide used for immunization or GST fusion proteins. Antibodies raised against Elfn1–GST and Elfn2–GST fusion proteins were further passed through an Elfn2–GST fusion protein-conjugated column and an Elfn1–GST fusion protein-conjugated column, respectively.

Animals

Elfn1-deficient mice were generated as described previously53. Briefly, to construct the Elfn1 targeting vector, overlapping Elfn1 genomic clones were obtained from BACPAC Resources Center at Children’s Hospital Oakland Research Institute (Oakland, CA). The targeting construct contained the 4.5-kb 5′ and 5.9-kb 3′ homology regions, and the 3.5-kb fragment containing the open reading frame of Elfn1 was replaced with the phosphoglycerol kinase (PGK)–neo expression cassette flanked by a loxP sequence. Embryonic stem cells (EmbryoMax Embryonic Stem Cell Line—Strain C57BL/6, Millipore) were electroporated with the targeting construct and selected with G418. Drug-resistant clones were analysed by Southern blotting. Chimeric mice were generated by injection of the targeted embryonic stem cells into BALB/c blastocysts. To excise the PGK–neo cassette, mice with germline transmission were first mated with transgenic mice expressing Cre recombinase in germline cells53. Correct excision of the PGK–neo cassette was confirmed by Southern blot. The resulting (Elfn1+/−, Cre-transgene) mice were backcrossed once to C57BL/6J mice to remove the Cre-transgenes. Elfn1+/– heterozygotes were used to generate Elfn1–/– mice, which are referred to as Elfn1 KO mice in this study. Genotyping was performed by Southern blot or PCR analysis of DNA isolated from tail samples. All experiments used male mice unless otherwise noted.

Immunoblotting

Regional and developmental specimens and subcellular fractions were prepared and subjected for IB as previously described53,54,55. For preparation of subcellular fractions, whole mouse brain was homogenized in buffered sucrose solution A (320 mM sucrose, 5 mM HEPES-KOH, pH 7.4) and centrifuged at 800g for 10 min to obtain S1 fraction. S1 fraction was further centrifuged at 10,000g for 30 min to obtain S2 fraction. S2 fraction was centrifuged at 100,000g for 1 h to obtain S3 fraction. P2 crude synaptosomal fraction was homogenized in solution A and layered on sucrose gradient (0.85 M, 1.0 M and 1.2 M sucrose containing 5 mM HEPES). After centrifugation at 100,000g for 2 h in a Beckman SW55Ti rotor, synaptosomal fraction was collected between 1.0 M and 1.2 M sucrose. Synaptosomal fraction was resuspended in buffered sucrose, lysed in 6 mM Tris–HCl (pH 8.0) and agitated at 4 °C for 45 min. LS1 and LP1 fraction was obtained after centrifugation at 32,800 × g for 20 min and LS1 fraction was further centrifuged at 165,000 g for 2 h to obtain LP2 pellet. LP1 pellet was resuspended in solution B (0.16 M sucrose in 6 mM Tris–HCl (pH 8.0) containing 1% Triton X-100 and protease inhibitor cocktail) and agitated at 4 °C for 15 min. After centrifugation at 32,800g for 20 min, the resultant pellet was resuspended in solution B and layered on sucrose gradient (1.0 M, 1.5 M and 2.0 M sucrose containing 5 mM HEPES). After centrifugation at 201,000g for 2 h in a Beckman SW55Ti rotor, synaptosomal plasma membrane and crude PSD fractions were collected above the 1.0 M layer of between 1.5 M and 2.0 M layer, respectively. Crude PSD fraction was resupended in modified solution B containing 75 mM KCl and agitated at 4 °C for 15 min. Purified PSD fraction was obtained after centrifugation at 201,000g for 20 min. Whole or parts of mouse brains from each developmental stage or region were homogenized in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and protease inhibitor cocktail. Protein extracts were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (Immobilon-P, Millipore) membranes. The membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween20 and probed with primary antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase. Blots were developed with ECL Plus reagents (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare).

Immunostaining for brain sections

Mice were anaesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). The brains were removed, postfixed for 3 h at 4 °C and sectioned at a thickness of 50 μm on a vibratome. Free-floating vibratome sections were incubated in blocking solution (PBS containing 10% normal goat serum and 0.1% Triton X-100) overnight, followed by incubation with primary antibodies for 3 days and with Alexa 488- or Alexa 594-conjugated secondary antibodies (Molecular Probes) overnight at 4 °C. To prepare the cryostat sections (12 μm), postfixed brains were cryoprotected in 20% sucrose (wt/vol) in PBS for 48 h at 4 °C, embedded in OCT compound and frozen on dry ice. For immunofluorescence staining, cryostat sections were incubated in blocking solution (PBS containing 10% normal goat serum and 0.1% Triton X-100) for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4 °C and with Alexa 488- or Alexa 594-conjugated secondary antibodies (Molecular Probes) for 1 h at room temperature. For cell counting analyses for SOM- or PV-positive interneurons, cryostat sections were autoclaved in 10 mM citrate buffer, pH 6.0, for 5 min at 105 °C for antigen retrieval (SOM) and incubated in 0.3% H 2 O 2 /methanol for 10 min. After incubation in blocking solution for 1 h at room temperature, sections were incubated with primary antibodies overnight at 4 °C and visualized by using the Vectastain ABC Elite system (Vector Laboratories). Direct labelling of the anti-Elfn1 antibody was carried out by using the Fluorescein labelling kit NH 2 (Dojindo). Immunostaining was performed at least three times with independent mouse brain sections and representative pictures were shown.

Primary neuronal cultures and immunostaining

Primary neuronal cultures were prepared from the hippocampi of (E) 18/19 Sprague–Dawley rats or (E) 16 mice. Briefly, the hippocampi were dissociated by treatment with trypsin EDTA and gently triturated with a pipette. The dissociated cells were plated onto poly-D-lysine-coated coverslips in Neurobasal medium supplemented with B27 (Invitrogen) and 2 mM L-glutamine. Cultured cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The frequency of the GAD67-positive cells in the GFP-positive transfectant was very low (0.3%) in our experimental system. For immunofluorescence staining of synaptic marker proteins, neurons were fixed with 4% PFA/4% sucrose in PBS on ice for 40 min or for 30 min followed by incubation in pre-chilled methanol for 10 min. Neurons were then permeabilized with Triton X-100, blocked with 5% normal goat serum and incubated with the primary and fluorescently labelled secondary antibodies. For mGluR7 staining, neurons were fixed with 100% methanol at −20 °C for 5 min and then incubated with primary and secondary antibodies in 30 mM phosphate buffer pH 7.4 containing 0.2% gelatin, 0.5% Triton X-100 and 0.8 M NaCl.

DNA constructs

Mouse Elfn1 cDNA was obtained through the FANTOM cDNA resource (http://fantom.gsc.riken.jp/). The open reading frame was PCR amplified and inserted into the pEF1-MycHis plasmid vector (Invitrogen). PCR-generated EGFP sequences that were in-frame with the Elfn1 sequence were inserted at an XbaI site in the C-terminal flanking sequence. To construct the Elfn1–Fc fusion protein expression vector, the PCR-generated Elfn1 ectodomain cDNA fragment was first inserted into the pAP-tag5 vector (GeneHunter). The resultant Elfn1 ectodomain sequence preceded by the immunoglobulin signal peptide sequence was PCR amplified and inserted into the pEF-Fc vector (a gift from Dr Yoshihiro Yoshihara, RIKEN BSI), allowing the target protein to be fused at the C terminus to the Fc region of human IgG1. The WT ELFN1 vector was generated by inserting human ELFN1 protein coding regions PCR amplified from the genomic DNA of a healthy human subject and cloned into the pGEMT easy plasmid vector (Promega). PCR-based site-directed mutagenesis was carried out by using appropriate primers for R650C, D678N and R691W. The resultant NotI fragments containing the entire protein coding region preceded by the Kozak consensus sequence were inserted into pCAG-ires-ECFP vectors in which ECFP was modified to have a GAP43-derived membrane anchor signal sequence. mGluR7 cDNA was PCR cloned by using Mouse 17-day Embryo Marathon-Ready cDNA (Clontech). The cDNA fragment was first inserted into the pAP-tag5 vector, and the intervening alkaline phosphatase sequence was removed by restriction endonuclease digestion, resulting in the mGluR7 ectodomain tagged at the C terminus with MycHis epitopes (pEF-mGluR7ecto-MycHis). The Flrt3 expression vector was constructed by PCR, using a mouse Flrt3 cDNA clone (FANTOM cDNA resource). The human SLITRK3 expression vector will be described elsewhere. The mGluR1aEGFP expression vector was provided by Dr Akihiro Mizutani (Showa Pharmaceutical University).

Synapse induction assay

Artificial synapse formation assays were performed with HEK293T cells as described56. The plasmids used in this experiment are pCMV5-NL1ΔAB EGFP, pCAG-Elfn1-iresEGFP, pCAG-Elfn2-iresEGFP and pCAG-iresEGFP alone.

mGluR7-recruiting activity assay

HEK293T cells were transfected with pCAG-ires-EGFP-based expression constructs by using TransIT LT1 reagent (Mirus Bio). After 48 h, cells were trypsinized, suspended in Neurobasal/B27 medium and laid onto the hippocampal neurons at DIV13 at a density of 1.5 × 104 cells. The co-cultured cells were maintained for 24 h, chilled on ice for 5 min and fixed with 4% PFA, 4% sucrose in PBS at 4 °C for 20 min. After four washes with PBS, the cells were immersed in a blocking buffer (5% normal goat serum, 3% bovine serum albumin, 0.1% Triton X-100 in PBS) and incubated with rabbit anti-mGluR7 and chicken anti-GFP diluted in the blocking buffer overnight at 4 °C. The bound antibodies were detected by Alexa 594-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-chicken IgY (Jackson Immunoresearch).

In vitro mGluR7-pull down binding assay

The Fc fusion protein was produced in HEK293T cells and affinity purified using protein G-Sepharose. HEK293T cells in a 10-cm dish were transfected with pEF-mGluR7ecto-MycHis. The cells were lysed in 1 ml of a buffer consisting of 0.5% Triton X-100, 150 mM NaCl and 50 mM Tris–HCl pH 7.5 with Complete Protease Inhibitor Cocktail (Roche). After incubation for 20 min at 4 °C with gentle rocking, the lysate was centrifuged at 13,000g for 20 min at 4 °C. For the binding assay, 50 μl of the supernatant was mixed with 950 μl of a binding buffer (10 mM HEPES, pH 7.4, 2 mM CaCl 2 , 1 mM MgCl 2 and 0.1% Triton X-100) containing 1.5 μg of Fc or Fc fusion proteins, and was incubated for 1 h at 4 °C with gentle agitation. After the binding reaction, the binding mixture was added by ImmunoPure Immobilized Protein G (Pierce), and further incubated for 1 h at 4 °C, followed by brief spinning. The proteins co-precipitated with the Protein G-resin were washed four times with the binding buffer and analysed by IB.

Imaging and quantitative immunofluorescence analysis

Image acquisition and analysis were performed by investigators blind to the experimental condition. For immunofluorescence experiment, single-plane images were captured using a FV1000 confocal microscope (Olympus) or BIOREVO BZ-9000 (Keyence) with identical settings. For transfected neurons, GFP- or CFP-expressing neurons were randomly chosen and imaged. The intensity values from the thresholded images were obtained using ImageJ software (http://rsb.info.nih.gov/ij/). For quantitative analysis of mGluR7 levels in brain sections, rectangular regions of interest were defined for CA1, SO and SR, and the intensity values from the thresholded images were obtained using ImageJ software. The regions of interest in CA1, OLM and DG hilus were determined from the mGluR1a-positive area. mGluR1a-positive spines were manually counted and normalized to 10-μm dendrite lengths. The length of the mGluR1a-positive structure was defined as the length from the dendrite to the tip of the mGluR1a-positive protrusions. For cell counting analysis of somatostatin- or PV-positive interneurons in the hp, images were acquired with a NanoZoomer slide scanner (Hamamatsu Photonics).

Immunoprecipitation

The crude synaptosomal (P2) fraction of mouse hippocampi was incubated in co-immunoprecipitation buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA) supplemented with protease inhibitor cocktail for 1 h at 4 °C. The lysates were obtained by centrifugation for 30 min at 10,000g, precleaned with anti-rabbit IgG agarose beads (Sigma), and incubated with 0.5 μg anti-Elfn1 or anti-mGluR7 antibodies. Complexes were isolated using anti-rabbit-IgG agarose beads.

Slice electrophysiology

Slices were prepared from adult (postnatal week 11–32) Elfn1 KO mice or their WT littermates by using the protective recovery method57. Transverse slices were used for recording pyramidal cells and coronal slices were used for recording OLM neurons. Mice were anaesthetized with isoflurane and sodium pentobarbital and transcardially perfused with a room temperature carbogenated (95% O 2 and 5% CO 2 ) protective artificial cerebrospinal fluid (ACSF) containing 92 mM N-methyl-D-glucamine, 2.5 mM KCl, 1.2 mM NaH 2 PO 4 , 30 mM NaHCO 3 , 20 mM HEPES, 25 mM glucose, 5 mM ascorbate, 3 mM sodium pyruvate, 12 mM N-acetyl-L-cysteine, 0.5 mM CaCl 2 and 10 mM MgSO 4 . The mice were then decapitated, the brains were removed and hippocampal slices were cut using a vibrating blade microtome (VT1200S, Leica) at room temperature. When cutting transverse hippocampal slices, the hp was dissected before slicing. The hippocampal slices were recovered in the carbogenated protective ACSF at 32 °C for 12 min and then transferred into a holding chamber containing a room temperature carbogenated incubating ACSF of the following composition: 119 mM NaCl, 2.5 mM KCl, 1 mM NaH 2 PO 4 , 26.2 mM NaHCO 3 , 12.5 mM glucose, 2 mM CaCl 2 ·4H 2 O, 2 mM MgSO 4 ·7H 2 O, 5 mM HEPES and 12 mM N-acetyl-L-cysteine.

After recovery for at least 2 h, the slices were transferred to a recording chamber mounted on an upright microscope (BX51WI, Olympus) equipped with IR-DIC optics. Slices were perfused with a carbogenated recording ACSF (at ~32 °C) containing 119 mM NaCl, 2.5 mM KCl, 4.0 mM CaCl 2 , 4.0 mM MgSO 4 , 1.0 mM NaH 2 PO 4 , 26.2 mM NaHCO 3 , 11 mM glucose, 5 mM HEPES and 0.1 mM picrotoxin. When measuring short-term facilitation of excitatory synapses onto OLM neurons, afferents in the SO were activated by a patch pipette that had a broken tip and was filled with recording ACSF, and EPSCs were recorded from OLM cells using a Multiclamp 700A (Molecular Devices). Recording patch electrodes were filled with an internal solution containing 135 mM caesium methanesulfonate, 10 mM HEPES, 0.2 mM EGTA, 8 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na 3 GTP and 0.4% biocytin (pH 7.2 with CsOH, osmolarity adjusted to 285 mOsm). Data acquisition and analysis were performed using custom Igor Pro (WaveMetrics) software routines. When analysing short-term facilitation in CA1 pyramidal neurons, another stimulating glass electrode was also placed in the stratum radiatum (two-pathway experiments). Perforated patch recordings were performed to study LTP of EPSPs in OLM neurons28. A stock of gramicidin (150 mg ml−1) in DMSO was prepared freshly every 2 h and diluted to 1:1,000 in a K+-based internal solution composed of the following: 135 mM K gluconate, 10 mM HEPES, 0.2 mM EGTA, 10 mM KCl, 4 mM Mg-ATP, 0.3 mM Na 3 GTP, 0.4% biocytin and 5 mM QX-314 Cl (pH 7.2 with KOH, osmolarity adjusted to 270 mOsm). The tip of a recording electrode was filled with a gramicidin-free internal solution and backfilled with a gramicidin-containing solution. Afferents to the OLM cell were activated every 20 s by a stimulating electrode placed in the SO, and baseline EPSPs were recorded in current-clamp mode for at least 10 min. To induce LTP, the amplifier was switched to voltage-clamp mode, neurons were held at −70 mV, and TBS(four bursts of five pulses at 100 Hz with an interval of 250 ms) was applied 10 times every 20 s28. After LTP was induced with TBS, the amplifier was returned to current-clamp mode, and EPSP recordings were continued. When the perforated patch recordings were finished, the membrane was ruptured by negative pressure or current injection and whole-cell mode was maintained for about 10 min to allow the OLM cells to be fully loaded with biocytin.

For post hoc identification of the recorded cells, slices were fixed overnight at 4 °C in a solution containing 4% PFA in phosphate buffer. The slices were then blocked with 2% normal donkey serum, 0.1% Triton X-100 in PBS for 1 h at room temperature, reacted with an anti-somatostatin antibody overnight, washed with PBS five times for 1 h each and reacted with an Alexa488-conjugated donkey anti-goat IgG antibody (Molecular Probes) and tetramethyl–rhodamine-conjugated streptavidin (Molecular Probes). Serial scans along the optical (z) axis were obtained for image analysis.

Handling-induced seizure susceptibility

Four to five mice were housed per cage and left undisturbed for 2 weeks before testing their susceptibility to seizures. On the day of testing, immediately after the cage was removed from the rack, each mouse was picked up by its tail and placed in a clean cage. The occurrence of seizure was determined by behavioural signs such as tonic–clonic convulsions, Straub tail, excessive salivation and loss of posture during the 2-min observation period.

Drug-induced seizure susceptibility

PTZ (Sigma) was dissolved in physiologic saline and administered intraperitoneally at a dose of 50 mg kg−1 to 6-month-old WT and KO mice. After the injection, each mouse was placed in a clear container and closely monitored for 10 min. An observer who was blind to the genotypes measured the latency to the first generalized seizure.

EEG recording

EEG recordings were performed with 7- to 8-month-old WT and KO mice as described previously58. Stainless steel screws (1.1 mm in diameter) that served as EEG electrodes were implanted over the somatosensory cortex (1.5 mm lateral to midline, 1.0 mm posterior to bregma) under 1.5% halothane anaesthesia with N 2 O:O 2 (3:2) ventilation 1 week before recording. A reference electrode was implanted on the cerebellum (at the midline, 2.0 mm posterior to lambda). Electromyography electrodes were placed 2–3 mm apart from the cervical region of the trapezius muscle. EEG and electromyography data were collected for 5 days.

Behavioural analysis

Behavioural analysis of adult WT and KO mice (4- to 5-month-old littermates) was performed as previously described59. Mice were single-housed 1 week before the start and during the behavioural analysis. The behavioural experiments were conducted during the light phase (10:00 hours to 19:00 hours). Experimenters were blind to genotype. Behavioural testing was performed in the following order: homecage activity test, open field test, light–dark box test, elevated plus maze test, startle response and prepulse inhibition test, hole board test, hot plate test, tail flick test.

Human subjects and mutation analysis of ELFN1

Each participating patient or the parent signed an informed consent form as approved by the Ethics Committees of RIKEN Institute and participating institutions. All of the patients and control individuals examined in this study are Japanese, a population that has been reported to have little or negligible stratification30,60,61. We first carried out a resequencing analysis of exons and their flanking intronic regions, using 184 patients with epilepsy (male, 99; female, 85; mean onset age 9.9 years; range, 2–21 years), 316 with autism (male, 262; female, 53; mean age 12.7 years; range, 3–32 years) and 311 control subjects (more than 20 years old). Genomic DNA from the peripheral venous blood of affected and unaffected individuals was extracted with a QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany). DNA for mutation screening was amplified with an illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare). We designed PCR primers to amplify all coding regions of ELFN1 (GenBank accession number, NM_001128636.2), and amplified genomic DNA by PCR with KOD-Plus (Toyobo). The PCR products were analysed by direct sequencing, using the ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems). All novel mutations identified in amplified DNA by GenomiPhi were verified by sequencing of PCR products, using genomic DNA as a template. Primer sequences and PCR conditions are available on request.

Missense mutations were found both in the patient group (9 single-nucleotide polymorphisms (SNPs)) and in controls (4 SNPs). Therefore, in a second step, we focused on 7 SNPs that were found only in the patient group (Supplementary Table 2). These missense mutations were further SNP-typed for 2170 control subjects (male, 889; female 1281; mean age 42.4 years; range, 14–86 years), and 4 (A481V, R650C, D678N, R691W) were finally identified as SNPs present only in patients in this study.

Statistics

No statistical methods were used to predetermine sample sizes. However, our sample sizes are similar to those reported in previous reports. Data distribution was assumed to be normal, although this was not formally tested. Statistical analyses were conducted by using the SPSS statistical package (ver. 16.0, SPSS Japan, Tokyo, Japan) or Statcel3 software (OMS, Saitama, Japan). Parametric data were analysed by using the two-tailed unpaired Student’s t-test or Welch’s t-test after testing for equality of variance by the F-test. Non-parametric data were analysed using Mann–Whitney’s U-test. Differences were defined as statistically significant when P<0.05.