Control vector c HA-Elfn2 HA- Control vector HA-Elfn2 HA- nti- nti-ha Hippocmpus (CA3) PV DAPI SOM DAPI Hippocmpus (dentte gyrus) PV DAPI SOM DAPI Supplementry Figure 1. Specificity of the nti- ntiody nd tissue distriution () Immunolot nlysis. N-terminlly HA-tgged or Elfn2 proteins were expressed in COS7 cells. The proteins in the cell lystes were detected y n ntiody, nd the sme lot ws re-proed with n nti-ha ntiody. This result indicted tht the ntiody used in this study does not cross-rect with Elfn2. () Immunofluorescence stining of in dult mouse rin coronl sections. spt, septum; d, digonl nd; mf, mgnocellulr sl forerin; lgp, lterl glous pllidus;, nterior mygdl; dhpc, dorsl hippocmpus; h, henul; sn, sustnti nigr; vhpc, ventrl hippocmpus; ip, interpedunculr nucleus; vtg, ventrl tegmentl nucleus. Scle r, 1 mm. (c) Immunostining showing clsses of -positive interneurons. (Top) Hippocmpus CA3 region. (Bottom) Hippocmpus dentte gyrus region. DAPI ws used s counterstin. Scle r, µm.
Fluorescence (Aritrry Units) Fluorescence (Aritrry Units) GAD67 GAD67 Control Neuroligin-1 Elfn2 HEK HEK HEK HEK c 4 3 2 1 Control Synpsin NL1 Elfn2 Control EGFP NL1 Elfn2.4.2 Control Rtio NL1 Elfn2 Supplementry Figure 2. Locliztion of endogenous proteins in inhiitory neurons nd rtificil synpse-formtion ssys demonstrting the lck of synptogenic ctivity () Dissocited hippocmpl neurons (DIV21) were immunostined for (green) nd GAD67 (mgent). ws detected in inhiitory neurons (closed rrowhed, GAD67-immunopositive som) ut not in excittory pyrmidl neurons (open rrowheds, GAD67-immunonegtive somt). Scle r, 2 µm. (, c) Artificil synpse-formtion ssys demonstrte tht nd Elfn2 hve no synptogenic ctivity. HEK293T cells expressing empty vector (Control), neuroligin-1-egfp (Neuroligin-1), nd EGFP (), or Elfn2 nd EGFP (Elfn2) were lid on top of hippocmpl neurons. After 2 dys of coculture, the cells were fixed nd stined for ntiodies ginst synpsin I nd EGFP. () Representtive imges. Red, Synpsin I; green, EGFP; yellow, overlp. (c) Quntifiction of synpsin I immunopositive signls overlpping with EGFP signls derived from HEK293 cells. Men vlues ± SEM re shown. P <.1, one-wy ANOVA followed y Tukey post hoc comprisons. n = 3 independent experiments. The dotted line represents the control vlue.
mglur1 mglur7 mglur1 mglur7 c Supplementry Figure 3. Immunostining of the trnsfected hippocmpl neurons () Dissocited hippocmpl neurons were trnsfected with -GFP t DIV7 nd immunostined t DIV9 for the excittory postsynptic mrker PSD95 (red) nd the excittory presynptic mrker Vglut1 (white) (upper pnel) nd mglur1 (red; ottom pnel). Arrowheds indicte positions of representtive Vglut1 punct. Scle rs, 1 µm. () Coexistence of endogenous mglur1 nd mglur7 proteins in cultured hippocmpl neurons t DIV17. Scle r, µm. (c) Dissocited hippocmpl neurons were trnsfected with ELFN1-iCFP t DIV7 nd immunostined for (red) nd the excittory presynptic mrker Vglut1 (white) t DIV9. Arrowheds indicte positions of Vglut1 punct. Scle rs, 1 µm.
5 11.1k Trgeting vector DTA DTA neo - 3 17.4k ORF 4.5k Neo Neo 3 proe 17.4k ORF proe d CX CB e IB: mglur1 mglur7 GluR6 PICK1 mido lck g mglur1 fluorescence intensity (% of ) f 11.1k 5 proe HP 25.k 17.4k 5.9k 25k c +/+ () +/-/- () Proes +/+ () +/-/- () + 12 8 6 4 2 OA SO SR CA1 hilus DG 9W OA SO SR CA1 hilus DG 4M Supplementry Figure 4. Trgeted disruption of the gene () Structures of the genomic locus, trgeting vector, nd mutted llele. Loctions of the 5, ORF, nd 3 proes for Southern lotting re shown. Open ox, protein coding region of the exon; gry ox, untrnslted region of the exon; Neo, neomycin-resistnce gene cssette; ORF, open reding frme; DTA, diphtheri toxin A; lck tringle, loxp site,, Eco site. Mice with germline trnsmission (neo) were crossed with CAG-Cre trnsgenic mice tht express Cre recominse in germline cells to delete the neo gene, creting. () Southern lot nlysis of Eco-digested genomic DNA using the 5, 3, nd ORF proes. (c) Immunolot nlysis of protein in hippocmpus (HP), cereellum (CB) nd cortex (CX) from dult nd mice. Asterisk indictes nonspecific nd. (d) Immunofluorescence stining using rin section from n mouse. Scle r, 5 mm. (e) Nissl stining of sgittl sections of dult nd mice showing norml gross morphology. Scle r, 2 mm. (f) Immunolot nlysis of the P2 frction from nd hippocmpi. Blots re representtive of two replicted experiments from independent mouse rin smples. (g) Quntifiction of the mglur1-immunopositive signls in hippocmpl suregions t 9 weeks (9W) nd 4 months (4M). DG, dentte gyrus; SO, strtum oriens; SR, strtum rditum. n = 4 (9W); n = 3 (4M) mice for ech genotype. Mesurements from two regions of interest, from right nd left hippocmpus, were verged per mouse.
mglur1 Vglut1 merge Vglut1 signl density neighoring mglur1 positive re (.u.) 7 6 4 3 2 1 merge mglur1 Vglut1 mglur7 Supplementry Figure 5. Vglut1 on mglur1-positive neurons () Quntifiction in sections. Left, CA1 strtum oriens from 1-week-old nd mice ws immunostined with nti-vglut1 (green) nd nti-mglur1 (red) ntiodies. Scle r, 1 µm. Grph t right, Vglut1-immunopositive signls on the mglur1-stined re were quntified (, n = 22 sections from 5 mice;, n = 27 sections from 6 mice; P =.63, unpired t-test). () Immunostining of cultured hippocmpl neurons. Hippocmpl neurons from nd emryos were cultured for 24 dys. The cells were stined with nti-mglur7 (green), nti-vglut1 (white) nd mglur1 (red) ntiodies, nd DAPI. Scle r, 2 µm.
Supplementry Figure 6. Developmentl profiles of nd mglur7 distriution Immunostining for nd mglur7 in emryonic dy 16 (E16), postntl dy (P) nd P3 hippocmpi (prsgittl sections). CA1, prospective CA1; CA3, prospective CA3; DG, prospective dentte gyrus; sterisks, CA1 CA3 prospective strtum oriens. Scle r, µm. DG
power mv^2/ Hz.8.6.4.2. 1 2 3 Hz power density (mv 2 /Hz) SWS REM W.8.6.4.2. W delt delt (1-4 Hz) SWS delt REM delt n.s. W delt SWS delt REM delt power mv^2/ Hz.8.6.4.2 SWS REM W. 1 2 3 Hz.5.4.3.2.1. W thet thet (6-9 Hz) SWS thet REM thet W thet n.s. SWS thet REM thet Numer of PV + cells (% of ) 14 12 8 6 4 2 CA1 CA3 DG CA1 CA3 DG Supplementry Figure 7. EEG spectrum nlysis () The EEG spectrum ws nlyzed y fst Fourier trnsform (4-s epochs during the drk period). The delt (1 4 Hz) nd thet (6 9 Hz) components were mesured in the wke (W), slow wve sleep (SWS) nd rpid eye movement (REM) phses. Men vlues ± SEM re shown., n = 3;, n = 3. n.s., not significnt, unpired t-test. () Prvlumin-positive interneurons. Prvlumin (PV)-positive cell numers were counted in the indicted 1W 4M hippocmpl suregions of immunostined sections. PV-positive interneuron numers did not differ significntly etween nd mice t 1 weeks (1W) or 4 months (4M). 1W,, n = 5; 1W, n = 6; 4M, n = 5; 4M, n = 4 mice.
Open field test Prepulse inhiition test Totl distnce (cm) 8 6 4 2 Totl center time (s) 3 2 Strtle response (ritrry units) 2 1.5 1.5 % PPI 8 6 4 2 PP7dB PPdB PP8dB c Til flick test d Hot plte test Ltency (s) 3 2 1 Ltency (s) 18 16 14 12 1 8 6 4 2 Lick Flinch Ltency (s) 9 6 3 Jump Supplementry Figure 8. Behviourl tests ( d) Adult nd mice (4 to 5 months old, n = 1 for ech genotype) were used for the ehviourl tests. Dt shown re mens ± SEM. nd mice did not differ significntly on the ehviourl tests shown.
Supplementry Figure 9-1 Full lots 1 IB: (G) IB: PSD-95 IB: SynPhy 1c IB: (G) IB: Actin 1d IB: (G) IB: Actin
2f IB:(C) IB:mGluR7 2h IB:Myc 25 25 2g IB: (G) IB: mglur7 193 113 96 53 36 Supplementry Figure 9-2 Full lots
Supplementry Figure 9-3 Full lots Suppl 4f mglur1 mglur7 GluR6 PICK1 21 115 96 52
Supplementry Tle 1 Numer of mice nlyzed for hndling-induced seizure frequencies None of the wild-type () nd heterozygous (Het) mice showed seizure-like ehviorl normlities in the experiment.
Supplementry Tle 2 Detils of the resequencing nlysis dsnp rs# cluster ID Function Gene position dsnp llele Amino cid position Protein residue rs182978 missense 289 G > A 97 Gly > Ser rs14423352 missense 566 G > A 189 Ser > Asn rs147351469 missense 74 G > C 235 Gly > Al frme shift 74 G > 235 Gly > Al Novel missense 77 G > A 236 Arg > His Novel missense 122 G > A 341 Arg > Gln Novel missense 1321 C > T 441 Arg > Cys rs148947783 missense 143 C > T 477 Al > Vl Novel missense 1442 C > T 481 Al > Vl Novel missense 146 C > T 487 Pro > Leu rs5869646 missense 1486 C > G 496 Arg > Gly rs1419181 missense 1888 G > A 63 Vl > Met Novel missense 1948 C > T 6 Arg >Cys Novel missense 232 G > A 678 Asp > Asn Novel missense 271 C > T 691 Arg > Trp Novel missense 2177 C > T 726 Pro > Leu Novel missense 2231 G > A 744 Arg > Gln rs111457141 missense 2357 G > A 786 Arg > Gln Novel missense 22 A > T 791 Glu > Vl Control (resequencing) Control (SNP typing) Epilepsy Autism G/G G/A A/A G/G G/A A/A 94 94 184 184 G/G G/A A/A G/G G/A A/A 94 94 184 184 G/G G/C C/C G/G G/C C/C 94 94 184 184 G/G G/- -/- G/G G/- -/- 94 94 184 184 G/G G/A A/A G/G G/A A/A G/G G/A A/A G/G G/A A/A 94 94 2166 4 217 184 184 315 1 316 G/G G/A A/A G/G G/A A/A G/G G/A A/A G/G G/A A/A 94 94 2169 1 217 184 184 315 1 316 C/C C/T T/T C/C C/T T/T C/C C/T T/T 31 1 311 184 184 316 316 C/C C/T T/T C/C C/T T/T 311 311 184 184 C/C C/T T/T C/C C/T T/T C/C C/T T/T C/C C/T T/T 311 311 2161 2161 184 184 315 1 316 C/C C/T T/T C/C C/T T/T C/C C/T T/T C/C C/T T/T 311 311 2164 1 2165 183 1 184 316 316 C/C C/G G/G C/C C/G G/G 311 311 184 184 G/G G/A A/A G/G G/A A/A 311 311 184 184 C/C C/T T/T C/C C/T T/T C/C C/T T/T C/C C/T T/T 311 311 216 216 184 184 315 1 316 G/G G/A A/A G/G G/A A/A G/G G/A A/A G/G G/A A/A 311 311 2169 2169 183 1 184 316 316 C/C C/T T/T C/C C/T T/T C/C C/T T/T C/C C/T T/T 311 311 2168 2168 183 1 184 316 316 C/C C/T T/T C/C C/T T/T C/C C/T T/T 31 1 311 184 184 315 1 316 G/G G/A A/A G/G G/A A/A G/G G/A A/A 31 1 311 184 184 316 316 G/G G/A A/A G/G G/A A/A 311 311 184 184 A/A A/T T/T A/A A/T T/T A/A A/T T/T 299 12 311 1 9 184 316 316 Detils for the epilepsy ptients Idiopthic epilepsy Symptomtic epilepsy JME 66 mtle 52 CAE 12 ICEGTC 7 JAE 1 TLE 3 GE 4 Epilepsy with myoclonic sence 2 GEFS+ 16 PE 1 Unclssified IGE 1 IGE with myoclonic seizures 1 Totl 119 65 JME: Juvenile myoclonic epilepsy JAE: Juvenile sence epilepsy CAE: Childhood sence epilepsy IGE: Idiopthic generlized epilepsy GEFS+: Generlized epilepsy with ferile seizures plus GE: Generlized epilepsy ICEGTC: Intrctle childhood epilepsy with GTC mtle: medil temporl loe epilepsy TLE: Temporl loe epilepsy PE: Prtil epilepsy