SIPA1L1/SPAR1 Interacts with the Neurabin Family of Proteins and is Involved in GPCR Signaling

Antibodies

Polyclonal anti-SIPA1L1, SIPA1L2, and SIPA1L3 antibodies were prepared by immunizing rabbits with fragments of SIPA1L1 (amino acids 1617–1804 of accession no. NP_056371.1), SIPA1L2 (amino acids 1–70 of accession no. NP_065859.3) or SIPA1L3 (amino acids 1049–1251 of accession no. NP_055888.1) fused to glutathione S-transferase. Antibodies were purified by affinity chromatography using columns to which antigens used for immunization had been linked. Other antibodies and reagents used in this work are listed in Table 1.

Targeted disruption of the Sipa1l1 gene

Genomic clones for the Sipa1l1 locus were isolated by screening with a C57BL/6N male liver genomic library (Clontech). The targeting vector was constructed by inserting a nuclear localization signal-LacZ-poly A cassette, followed by a PGK-neo-poly A cassette at the first methionine site, preserving 0.8 kb (5′) and 5.2 kb (3′) of the flanking regions (Fig. 1A). TT2 ES cells were electroporated and selected by standard procedures. Correctly targeted clones were screened by PCR and subsequently confirmed by Southern blotting. Targeted clones were used for aggregation with eight-cell embryos, and chimeric males were mated with C57BL/6N females. Subsequent genotyping was performed by genomic PCR. Primers used were forward: 5′-TAGATCCGTGTGCCACAA-3′, reverse: 5′-GAGGCCAATCTGCTATTC-3′, and LacZ: 5′-CAGTCACGACGTTGTAAAAC-3′. Heterozygotes were then backcrossed to C57BL/6N mice for at least nine generations. Two- to four-month-old male mice were used for all experiments. They were kept on a 14/10 h light/dark cycle in a temperature-controlled and humidity-controlled (22–24°C, 50–60%) specific pathogen-free vivarium, and they had ad libitum access to food and water. All animal experiments were conducted according to guidelines for care and use of animals, approved by the Animal Experiment Committee of the Institute for Quantitative Biosciences, The University of Tokyo and the Okinawa Institute of Science and Technology. License numbers for animal experiments are 23008, 24008, 2514, 2604, 2703, 2801 for the University of Tokyo and 2016-139, 2019-236 for the Okinawa Institute of Science and Technology.

Immunohistochemistry and X-Gal staining

Mice were deeply anesthetized with 90 mg/kg sodium pentobarbital and were intracardially perfused with ice-cold sodium phosphate buffer (pH 7.3, NPB), followed by ice-cold 4% PFA/NPB. Whole brains were removed, separated bilaterally at the medial line, and fixed in ice-cold 4% PFA/NPB for 2 h. Brains were further infiltrated sequentially with 10%, 15%, and 20% sucrose/NPB for >4 h at each concentration and then frozen in a Tissue-Tek OCT compound (Sakura Finetek); 10-µm cryosections were attached to an MAS-coated glass slide (S9441 Matsunami) and air dried for 2 h. For permeabilization, sections were incubated in 0.3% Triton X-100/Tris-buffered saline (pH 7.5; TBS) for 10 min at room temperature (RT) except for synaptophysin and spinophilin (mouse antibody) staining. Alternatively, sections were incubated in 0.4 mg/ml pepsin in 0.2 N HCl for 2 min at 37°C for a PSD localization assay. For synaptophysin and spinophilin (mouse antibody) staining, sections were incubated in boiling 10 mm sodium citrate for 5 min. Sections were blocked with TBS containing a 0.5% blocking reagent (Roche), 2% fetal bovine serum, and 0.1% Tween 20 for 1 h. Then they were incubated overnight at 4°C with primary antibodies diluted in the blocking buffer. In the case of reactions containing mouse antibodies, reagents from the VECTOR M.O.M. Basic kit (Vector Laboratories) were added. After washes in TBS containing 0.1% Tween 20 (TBST), sections were incubated for 1 h at RT with secondary antibodies diluted in blocking buffer. Sections were subsequently stained with TOPRO-3 or DAPI, washed, and coverslipped with Vectashield mounting medium (Vector Laboratories). Sections from wild-type (WT) and knock-out (KO) mice were processed simultaneously on the same slide glass. Antibodies and their dilutions used for immunostaining are listed in Table 1. A Zenon Rabbit IgG Labeling kit (Invitrogen) was used for staining neurabin-1 according to manufacturer's instructions. Briefly, labeling of SIPA1L1 was performed following a regular protocol as above until secondary antibody incubation. After TBST washes, sections were incubated with Zenon-labeled (molar ratio 6:1) neurabin-1 antibody for 1.5 h at RT. After three TBST and two PBS washes, sections were fixed with 4% PFA for 15 min, washed, and coverslipped with Vectashield mounting medium. A parallel experiment using negative-control omitting neurabin-1 antibody in the Zenon labeling reaction resulted in no significant signal. For diaminobenzidine (DAB) staining, Vectastain Elite ABC and Avidin/Biotin Blocking kits (Vector Laboratories) were used in conjunction with the above procedures. For X-Gal staining, dried sections were stained overnight at 37°C in an X-Gal staining solution and subsequently counterstained with Nuclear Fast Red (Vector Laboratories). Nissl staining was performed by incubating sections in a 1% thionin solution for 60 min at RT. Digital images were obtained using commercial Olympus microscopes. Briefly, low magnification images were obtained with an IX-83 (Olympus) equipped with a DP80 camera and a motorized stage using a 20× objective. Whole-brain sections or regions of interest were scanned and stitched automatically using cellSense software (Olympus). For confocal imaging, either an FV1000 (Olympus) with a 100×, 1.4 NA silicone immersion objective UPLSAPO100XO or a Leica TCS SP8 LIGHTNING confocal microscope equipped with an HC PL APO CS2 20×/0.75 DRY objective, motorized stage, and LAS X software was used. For SRM, the SD-OSR (Olympus) equipped with a Yokogawa CSU-W1 scanner, Hamamatsu Orca Flash 4 V2+ High Speed SCMOS camera, and a 100×, 1.35 NA silicone immersion objective with correction collar (UPLSAPO100XS) was used to acquire Z-stack images (200-nm step size, all channels scanned in each plane) of 1024 × 1024 pixels (41 × 41 µm²)/image. Original images adjusted only for brightness and contrast with Fiji/ImageJ (NIH) are shown in the figures.

IEM

Mice were fixed by transcardial perfusion with 3% glyoxal-based fixative (Richter et al., 2018). Brains were cryoprotected with 30% sucrose in 0.1 m phosphate buffer to prepare 50-µm cryosections on a cryostat (CM1900; Leica Microsystems). All immunohistochemical incubations were performed at RT. For silver-enhanced preembedding immunogold electron microscopy, microslicer sections were dipped in 10% normal goat serum /PBS for 30 min, incubated overnight with SIPA1L1 antibody (1:1000) diluted with 0.1% Triton X-100/PBS, and subjected to silver-enhanced immunogold labeling using anti-rabbit IgG conjugated with 1.4-nm gold particles (Nanogold) and R-Gent SE-EM Silver Enhancement Reagents (Aurion). Sections were further treated with 1% osmium tetroxide and 2% uranyl acetate, and embedded in Epon812. Ultrathin sections (100 nm) were prepared with an ultramicrotome (Leica), and photographs were taken with an H7100 electron microscope (Hitachi). The distribution of immunogold particles was quantitatively analyzed on electron micrographs using MetaMorph software (Mlecular Devices; n = 2 mice). Perpendicular distribution of PSD-95 or SIPA1L1 was examined by sampling synaptic profiles whose presynaptic and postsynaptic membranes were cut perpendicularly to the plane of the synaptic cleft, and by measuring the distance from the midline of the synaptic cleft to the center of immunogold particles. Statistical significance was assessed using the Kolmogorov–Smirnov test.

Spine size analysis by electron microscopy

Electron microscopy was basically performed as described (Meng et al., 2002). Briefly, littermate mice (two to three months) were deeply anesthetized with sodium pentobarbital and were intracardially perfused for 5 min with 2.5% glutaraldehyde in a 0.1 m phosphate buffer (pH 7.4). Hippocampi were removed from whole brains, and the CA1 areas of hippocampi were cut into tiny blocks. These blocks were postfixed in the same fixative for 3 h, osmicated with 1% osmium tetroxide in a 0.1 m phosphate buffer for 2 h, washed thoroughly with 5% sucrose, dehydrated in a graded alcohol series, embedded in Epok812 (#02-1001, Okenshoji Co), and cured for 12 h at 60°C. For each block, 1-μm sections were cut and stained with 1% toluidine blue to guide further trimming to isolate the equivalent CA1 subfields. Ultrathin sections (80 nm) were cut with a diamond knife and stained with uranyl acetate and lead citrate, and then observed with a JEOL JEM1010 electron microscope operated at 100 kV. Similar neuropil areas of the stratum radiatum not containing cell bodies or blood vessels were randomly selected within 100–250 μm of the CA1 pyramidal cell body and photomicrographed at 5000× magnification. Five electron micrographs representing 1300- to 1400-μm² neuropil regions of each mouse were taken. Image negatives were scanned at 1200 dpi and analyzed with ImageJ. The number of synapses (synapse density), PSD lengths, and cross-sectional areas of spine heads from four mice per genotype were quantified. Excitatory synapses bearing spines were defined by the presence of a clear PSD facing at least three presynaptic vesicles. Measurements were performed by an experimenter blind to the genotype.

Electrophysiological analysis

Standard procedures and solutions described previously (Kobayashi et al., 2016) were used. In brief, hippocampal slices (400 µm) were prepared from mice 8–12 weeks of age. Synaptic responses were recorded at 25.0 ± 0.5°C with extracellular field-potential recordings in the stratum radiatum of the CA1 region using a glass recording pipette filled with 3 m NaCl. External solution contained the following: 119 mm NaCl, 2.5 mm KCl, 1.3 mm MgSO₄, 2.5 mm CaCl₂, 1.0 mm NaH₂PO₄, 26.2 mm NaHCO₃, 11 mm glucose, and 0.1 mm picrotoxin (a GABA_A-receptor antagonist). To evoke synaptic responses, Schaffer collateral/commissural fibers were stimulated at 0.1 Hz (test pulse) with a bipolar tungsten electrode. Stimulus strength was adjusted to evoke EPSPs with a slope of 0.10–0.15 mV/ms, except for experiments examining input-output relationships. Input–output relationships were examined in the presence of a low concentration of the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX: 1 μm) in the external solution to partially block AMPA receptor-mediated EPSPs. This partial blockade enables more accurate measurements since the nonlinear summation of field EPSPs is reduced. EPSPs were evoked with various strengths of stimulation, and data were first sorted by binning fiber volley amplitudes. Then EPSP amplitudes were averaged within each bin. Paired-pulse facilitation was examined in the presence of 25 μm D-(–)−2-amino-5-phosphonopentanoic acid (D-AP5). Paired-pulse stimuli at intervals of 50, 100, and 200 ms were applied every 10 s. An Axopatch-1D amplifier (Molecular Devices) was used to record EPSPs. Data were digitized at 10 kHz and analyzed on-line using pClamp software (Molecular Devices). All values were reported as mean ± SEM. Student's t test was used to determine whether there was a significant difference in the means of two datasets. Picrotoxin was purchased from Sigma-Aldrich. D-AP5 and CNQX were purchased from Tocris Bioscience.

Colocalization analysis

Colocalization analysis was performed using GDSC ImageJ plug-in according to the developer's Colocalization User Manual (http://www.sussex.ac.uk/gdsc/intranet/microscopy/UserSupport/AnalysisProtocol/imagej/colocalisation). Briefly, SRM images stained for SIPA1L1 and its candidate interacting-proteins at the neuropil region of Layer V cerebral cortex or hippocampal CA1 area were acquired using Olympus SD-OSR as described above. Four serial Z-stack images (200-nm step size) of 1024 × 1024 pixels (41 × 41 µm²)/image were processed to define foreground and background using the Otsu method (or the Triangle method for images with relatively low signal-to-noise ratio) using Stack Threshold Plugin. Processed images and raw images were used to calculate the statistical significance of Manders coefficient and Pearson correlation coefficient, respectively, with the confined displacement algorithm Plugin. If the presence of irregular structures such as somata interfered with the analysis, the corresponding region was excluded from the confined region. Random displacement was defined using a radial displacement chart for Pearson correlation coefficient for each sample. A P value of <0.01 was adopted for statistical significance. Raw image data used in the analyses were deposited on Mendeley at https://doi.org/10.17632/f964whtpxh.1.

Cell culture, transfections, immunostaining, and immunoprecipitation (IP)

HEK293T or COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C, 5% CO₂. Expression vectors for the SIPA1L family of proteins were generated by cloning human SIPA1L1 (NM_015556.3), SIPA1L2 (AY168879), or SIPA1L3 (AY168880) cDNA into FLAG- or Myc-pcDNA3.1 (+). Expression vectors for spinophilin have been described elsewhere (Sagara et al., 2009). Transfections of plasmid constructs were performed using Lipofectamine 2000 (Invitrogen) for HEK293T and Lipofectamine LTX (Invitrogen) or TransIT-LT1 (Mirus Bio) for COS-7 cells, according to manufacturers' instructions. Immunostaining was performed as described previously (Sagara et al., 2009). For IP, COS-7 cells were lysed in lysis buffer (0.33% SDS, 1.67% Triton X-100, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 1 mm Na₃VO₄, 25 mm NaF, 5 µg/ml aprotinin, chymostatin, leupeptin and pepstatin A, 10% glycerol), rotated for 60 min at 4°C and centrifuged at 17,000 × g for 40 min. Supernatants were precleared with Dynabeads Protein G (Invitrogen Dynal) and incubated with anti-Myc (MBL) antibody-bound Dynabeads Protein G with overnight rotation at 4°C. Samples were then washed four times with wash buffer (0.33% SDS, 1.67% Triton X-100, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol). Proteins were eluted by incubation in 50 mm Tris, pH 6.8 with 2% SDS for 10 min at RT with shaking. Samples were analyzed by Western blotting (WB), as above.

Crosslinking IP combined with mass spectrometry (cIP-MS) and cIP-Western blotting (cIP-WB) analyses

Mouse brain regions of interest were quickly dissected on a filter paper soaked with ice-cold homogenization buffer (HB; 0.32 m sucrose, 20 mm HEPES pH 7.4, 1 mm EDTA, 1 mm EGTA, 5 mm NaF, and 1 mm Na₃VO₄) and were homogenized in ice-cold HB using a Dounce homogenizer. After homogenates were centrifuged at 800 × g for 10 min, supernatants were transferred to new tubes, and proteins were crosslinked by adding 20 mm DSP (dithiobis [succinimidylpropionate], Thermo Scientific Pierce), a primary amine-reactive and membrane-permeable crosslinker with a 1.2 nm spacer arm, to a final concentration of 200 μm. For noncrosslinked controls, the same volume of solvent (DMSO) was added. Tubes were rotated at 4°C for 10 min, and 1 m Tris-Cl (pH 7.4) was added to a final concentration of 100 mm to terminate the crosslinking reaction. After 15 min of rotation, tubes were centrifuged at 9200 × g to obtain the P2 fractions, containing crude synaptosomes and plasma membranes. P2 pellets were solubilized in lysis buffer (2% SDS, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 1 mm Na₃VO₄, 25 mm NaF, 5 µg/ml aprotinin, chymostatin, leupeptin and pepstatin A, 10% glycerol) at 37°C for 30 min. Five times the volume of ice-cold neutralization buffer (2% Triton X-100, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 1 mm Na₃VO₄, 25 mm NaF, 5 µg/ml aprotinin, chymostatin, leupeptin and pepstatin A, 10% glycerol) was added and centrifuged at 17 000 × g for 1 h. Supernatants were precleared using Dynabeads Protein G (Invitrogen Dynal) and incubated with anti-SIPA1L1, anti-PSD-95 (Millipore Biotechnology), or control IgG antibody with overnight rotation at 4°C. Samples were then rotated with Dynabeads Protein G for 90 min and washed four times with wash buffer (0.33% SDS, 1.67% Triton X-100, 50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol). Proteins were decrosslinked and eluted by incubation in a 2× SDS sample buffer containing 200 mm DTT for 60 min at 37°C followed by 10 min at 56°C with constant mixing at 1400 rpm on an Eppendorf ThermoMixer. Dynabeads and unsolubilized materials were carefully removed magnetically and by centrifugation. Final supernatants were analyzed by SDS-PAGE.

For silver staining analysis, Perfect NT Gel (5–20%, DRC Co) was used for SDS-PAGE. Staining and destaining were performed using SilverQuest (Invitrogen), according to the manufacturer's protocol. Selected bands or corresponding areas in control lanes were excised and cut into 1 mm cubes, destained, and reduced with DTT (10 mm in 100 mm NH₄HCO₃, 56°C for 60 min) followed by alkylation with iodoacetamide (55 mm in 100 mm NH₄HCO₃ RT for 45 min). After repeated alternate washings with 100 mm NH₄HCO₃ and acetonitrile, gel pieces were rehydrated with 10 µl 50 mm NH₄HCO₃ containing 25 µg/ml trypsin (Trypsin Gold, Promega) and incubated for 15 min on ice; 10 µl of 50 mm NH₄HCO₃ was added, and trypsin digestion was conducted overnight at 37°C. Peptides were extracted with 20 µL of 20 mm NH₄HCO₃, followed by 20 µl of 50% acetonitrile, 0.1% trifluoroacetic acid (TFA) three times. The volume of pooled supernatants was reduced to 10–20 µl by vacuum centrifugation and then loaded into an automated electrospray ionization (ESI)-MS/MS system, which consisted of the DiNa system (KYA Tech Corporation) equipped with a C18 ESI capillary column (100 μm × 150 mm, NIKKYO Technos) and an LTQ Velos Orbitrap ETD instrument (ThermoFischer Scientific). For protein identification, spectra were processed using Proteome Discoverer Version 1.2 (ThermoFisher Scientific) against SEQUEST with a 5% false discovery rate (FDR) cutoff. Experiments were performed once each for cerebral cortex and hippocampus. Candidate SIPA1L1-interacting proteins were defined as proteins detected only in WT samples (PSMs ≧ 2) or if PSMs of WT were >10-fold of that of KO samples.

WB was performed by standard methods. Briefly, proteins were transferred to PVDF membranes (Immobilon, Millipore) and 5% skim milk, 0.1% Tween 20 in TBS was used for blocking and antibody dilution. Antibodies and associated dilution factors are listed in Table 1. A chemiluminescent signal was detected using Luminata Forte Western HRP (Millipore) and ImageQuant LAS4000mini (FujiFilm)

GPCR agonist stimulation (sedation) analysis

Nine- to 10-week-old mice were evaluated in the rotarod test for sedation, basically as described previously (Wang et al., 2004). Briefly, the subject was placed on a rotarod (O'hara & Co) rotating constantly at 10 rpm. Mice were trained for three to six sessions until they learned to remain on the rod for 60 s. Mice were injected intraperitoneally with increasing doses of the α₂AR-agonist, UK 14304 (abcam), or the adenosine analog, (-)-N6-(2-Phenylisopropyl) adenosine (R-PIA; Sigma-Aldrich) dissolved in saline. Ten minutes after injection, each mouse was tested three times in succession for its ability to remain on the rotarod. Results of the three trials were averaged. Cumulative doses of agonists are shown in the figure. The cutoff time was 60 s. The experimenter was blinded to the genotype during testing.

Seizure susceptibility analysis

Eight- to nine-week-old mice were injected intraperitoneally with 30 mg/kg of kainate (Sigma-Aldrich) or pentylenetetrazole (PTZ; Sigma-Aldrich) dissolved in saline in a volume of 15 ml/kg. For the α₂AR agonist treatment, eight- to nine-week-old mice were injected intraperitoneally with 1 mg/kg of guanfacine (Sigma-Aldrich) dissolved in saline in a volume of 6 ml/kg 30 min before the injection of PTZ. Mice were placed in a clear Plexiglas cage and videorecorded for up to 2 h or the 30-min cutoff time for kainate-induced or PTZ-induced seizures, respectively. Seizures were scored according to the following scale. Phase 1, hypoactivity: a progressive decrease in motor activity until the animal came to rest in a crouched or prone position with the abdomen in full contact with the cage; phase 2, partial clonus: a brief seizure, typically lasting 1 or 2 s, with clonic seizure activity affecting the face, head or forelimbs; phase 3, generalized clonus: the sudden loss of upright posture, whole body clonus involving all four limbs and tail, typically lasting for 30–60 s, followed by a quiescent period; and phase 4, severe generalized tonic-clonic seizure: a continuous loss of upright posture, lying or rolling on the floor, resulting in death from continuous convulsions. When a phase was skipped, the same latency of higher phases was also adopted for the lower phase. The experimenter was blinded to the genotype during testing.

Behavioral analysis

Male Sipa1l1^−/− and WT mice were housed together, with two to four littermates (or mice with close birthdays) per cage after weaning. Mice were acclimated to handling and the experimental room for at least 3 d before the start of an experiment. An independent group of mice (two to three months) was used for each test unless otherwise noted. Experimenters were blinded to the genotype during testing. All experiments were analyzed using an automated system from O'hara & Co, except during eyeblink conditioning. All Image series software (O'hara & Co) used for analysis is based on the public domain NIH Image or ImageJ program (https://imagej.nih.gov/nih-image/).

Open field test

Each subject was placed in the center of an open-field apparatus (50 × 50 × 33.3 cm; width × depth × height) illuminated at 20 lux and allowed to move freely for 10 min. Distance traveled in the arena, trace of the movement, rearing activity, and time spent in the center were recorded and analyzed using Image OF 2.15× and Image OFC 2.03sx. Rearing activity was counted manually using the human observation mode of Image OFC 2.03sx. Accelerating rotarod and contextual and cued fear conditioning tests were subsequently performed on the same group of mice with an interval of 2 d between tests.

Accelerating rotarod test

Mice were placed on a rod (3 cm in diameter) rotating at 4 rpm initially, and then the rotation of the rotarod was accelerated linearly to 40 rpm over a 300-s period. Latency to fall off the rotarod during a trial was automatically measured. Mice were trained for two consecutive days, receiving three trials per day at intervals of 90 min between trials.

Light-dark transition test

The apparatus consisted of a box (21 × 42 × 25 cm) divided into two sections of equal size by a partition with a door. One chamber was brightly illuminated (100 lux), whereas the other chamber was dark without illumination. Mice were placed on the dark side and allowed to move freely between the two chambers with the door open for 10 min. The total number of transitions, time spent on each side, latency to enter the light side, and distance traveled were recorded and analyzed automatically using ImageJ LD1.

Morris water maze

A pool 1 m in diameter was filled with opaque water colored with nontoxic white paint and maintained at ∼25°C. Each training trial began by placing the mouse in the quadrant that was either right, left, or opposite to the target quadrant containing a submerged platform (10 cm in diameter), in semi-random order. The same order of start positions was used for all subjects. Training trials were a maximum 60 s in duration. A mouse that failed to reach the platform within 60 s was subsequently guided to the platform. Mice that reached or were guided to the platform stayed there for 20 s. Two trials per block with a 1-min intertrial interval, three blocks per day with a 1-h interblock interval were conducted for 10 or 5 d to train mice for hidden or visible platform tasks, respectively. The visible platform test was conducted after completion of the hidden platform test. Latency to reach the platform, distance traveled to the platform, and average swim speed were automatically recorded. At the end of the tenth day of hidden platform training, a probe test was conducted for 1 min to confirm that spatial learning had been acquired, based on navigation by distal environmental room cues. Time spent in each quadrant and the number of crossings above the original platform site were automatically recorded. Data were automatically analyzed using Image WM 2.12r, Image WMV 2.08 sr, and Image WMH 2.08s.

Three-chambered social interaction test

Four-month-old mice were used for this social interaction test. The testing apparatus consisted of a rectangular, three-chambered box and a lid with an LED light panel and a CCD monochrome camera. Each chamber was 20 × 40 × 22 cm, and separating walls were made from transparent Plexiglas with small openings (5 × 3 × 3 cm). The subject mouse was first placed in the middle chamber and allowed to habituate to the entire test box for 10 min. After habituation, the mouse was taken out of the box, and an age-matched unfamiliar WT male (stranger mouse), which had no prior contact with the subject mouse, was placed in a small, round wire cage in one of the side chambers. The side on which the stranger mouse was placed was systematically alternated between trials. The subject was placed back in the central chamber for a 10-min session, and the time spent in each chamber, the number of entrances to each chamber, the distance traveled in each chamber or in the periphery of each cage, the total distance traveled, the average travel speed, and the heatmaps were automatically recorded and analyzed using TimeCS1 software (O'hara & Co).

Eyeblink conditioning

Mice were prepared for eyeblink conditioning basically according to previously described procedures (Takatsuki et al., 2003). In brief, under anesthesia with pentobarbital and, if necessary, with diethyl ether inhalation, four Teflon-coated stainless-steel wires (No. 7910, AM Systems) were implanted under the left upper eyelid. Two of these wires were used to record the eyelid electromyograms (EMGs), and the remaining two delivered an unconditioned stimulus (US). Two to three days after surgery, mice were subjected to 2 d of habituation without US or conditioned stimulus (CS), during which EMGs were recorded to calculate spontaneous eyeblink frequency. Seven or 10 d of delay or trace conditioning, respectively, began the next day. A daily session consisted of 90 CS-US paired trials and 10 CS-alone trials at every 10th trial. The CS was a 350-ms tone (1 kHz, 90 dB) with a 5-ms rise and a 5-ms fall time. The US was a 100-ms periorbital shock (100-Hz square pulses) with the intensity carefully adjusted to elicit a head-jerk response in each animal. The interstimulus interval was 250 or 850 ms in delay or trace conditioning, respectively. Eyelid EMGs were analyzed as described previously (Takatsuki et al., 2003), except that trials that elicited a startle response to the CS were also included for evaluation of conditioned response (CR) occurrence. In brief, the mean ± SD of amplitudes of EMG activity for 300 ms before CS onset in 100 trials was defined as the threshold, which was then used in the analysis below. In each trial, average values of EMG amplitude above the threshold were calculated for 300 ms before CS onset (prevalue), 30 ms after CS onset (startle-value), and 200 ms before US onset (CR-value). If the prevalue was <10% of threshold, the trial was regarded as valid. Among valid trials, a trial was assumed to contain the CR if the CR value was larger than 1% of the threshold and it exceeded two times the prevalue. For CS-alone trials, the period for CR-value calculation was extended to the CS end. To evaluate the effect on the startle response, we calculated the frequency of trials in which the startle-value exceeded 10% of the threshold.

Experimental design and statistical analyses

All results are expressed as mean ± SEM, unless noted otherwise. Statistical analyses in this work employed unpaired two-tailed Student's t tests, two-tailed Welch's t tests, two-tailed Mann–Whitney tests, two-tailed Wilcoxon matched pair signed-rank tests, Kolmogorov–Smirnov tests, one-way ANOVA with Geisser–Greenhouse correction followed by Tukey's post hoc tests, or a two-way ANOVA with Geisser–Greenhouse correction followed by Sidak's post hoc tests, where appropriate, using GraphPad Prism 8. A P value of <0.05 was considered statistically significant. For effect size calculations, Pearson's r or partial η² was used. D'Agostino–Pearson test and F tests were used to check normality and equal variance, respectively. More statistical information is available in Extended Data Figure 18-1.

Availability of data and materials

Most of the data generated or analyzed during this study are included in this article and its supplementary information files. Other datasets generated during and/or analyzed during the current study are available from the corresponding authors on request. Most materials are readily available from commercial sources or from our lab. Exceptions are the rabbit polyclonal anti-SIPA1L1, 2, or three antibodies that we generated, or antibodies discontinued from commercial suppliers, because of limited amounts. However, they may be made available for reasonable request.