Experimental subjects

Nlgn2 KO mice21 were generated in our laboratory on an 129/Sv background and were backcrossed onto a C57BL/6 J background for at least six generations. IgSF9b KO mice were obtained from Lexicon Pharmaceuticals (The Woodlands, TX, U.S.A.; Omnibank clone 281214, generated through insertion of the Omnibank gene trap vector 48 into the IgSF9b gene in Sv129 ES cells) and were backcrossed onto a C57BL/6 J background for at least six generations. Nlgn2 KO mice and IgSF9b KO mice were crossed to generate Nlgn2 het x IgSF9b het breeding pairs, from which four experimental genotypes were obtained as littermates, i.e., WT, Nlgn2 KO, IgSF9b KO, and double KO mice. For in vitro electrophysiology experiments, these mice were additionally crossed with mice expressing a Venus transgene under the control of the VIAAT promoter to label inhibitory interneurons (generated using a Venus construct generously provided by Dr. Atsushi Miyawaki, RIKEN)32. All mice were 2–3 months old at the beginning of the experiment. For experiments involving OF testing (Figs. 1–4 and Supplementary Fig. 1–4), only male mice were used unless specified otherwise. For the EPM experiment (Fig. 1h–m) and for molecular and slice electrophysiology experiments (Figs. 5–7), both male and female mice were used, based on the observation that the OF phenotype is identical in both sexes. Animals were maintained on a 12 h light/dark cycle (7 am/7 pm), with food and water ad libitum. All experiments were performed during the light cycle (with the exception of home cage activity monitoring as described below, Supplementary Fig. 2). The experimenter was blind to genotype during all stages of data acquisition and analysis. All procedures were approved by the State of Niedersachsen (Landesamt für Verbraucherschutz und Lebensmittelsicherheit) and were carried out in agreement with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany and the Max Planck Society.

Behavioral characterization

The OF test was conducted in a square arena made of white plastic (50 cm × 50 cm). Mice were placed in the corner of the OF and were permitted to explore the arena for 10 min. The EPM was conducted in an apparatus made of gray plastic, with two open arms and two closed arms (28 cm × 5 cm each, elevated 45 cm from the ground). Mice were placed in the closed arm and were permitted to explore the apparatus for 5 min. Performance was recorded using an overhead camera system and scored automatically using the Viewer software (Biobserve, St. Augustin, Germany). Between each mouse, the arena was cleaned thoroughly with 70% ethanol followed by water to eliminate any odors left by the previous mouse.

In our previous study8, the center zone of the OF was defined as a square of 25 × 25 cm. However, closer assessment revealed that a second, intermediate zone (12.5 cm–25 cm from the walls of the chamber) was also avoided by the Nlgn2 KO mice (Supplementary Fig. 1a-h), and that in fact the behavior in this intermediate zone resembled the behavior in the center, rather than the periphery, in all four genotypes assessed in the present study. For this reason, an extended center zone of 37.5 × 37.5 cm was used throughout this study to more accurately reflect the anxiogenic area.

Recording of home cage activity was performed using the LABORAS system and software (Metris, Hoofddorp, The Netherlands). Mice were habituated to the LABORAS cages for two days. On the third day, activity was recorded for two 6-hour periods, from 9 pm to 3 am (dark cycle) and from 9 am to 3 pm (light cycle). The following parameters were assessed: total duration of locomotor activity, immobility, grooming, and climbing, as well total distance traveled and average velocity during locomotion.

cFos induction assay

To assess anxiety-induced cFos activation, mice were first exposed to the OF arena for 10 min. Ninety minutes after exposure, they were anesthetized with Avertin (Tribromoethanol, Sigma) and perfused transcardially first with saline, then with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Brains were post-fixed in PFA overnight, and cryoprotected in 30% sucrose in 0.1 M PB. Free-floating sections (40 μm thickness) were prepared using a Leica CM3050S cryostat (Leica, Wetzlar, Germany). Sections were incubated in blocking solution (3% bovine serum albumin, 10% goat serum, 3% Triton X-100 in 0.1 M phosphate-buffered saline (PBS)) for 1 h, then incubated for 12 h with Rabbit polyclonal anti-cFos antibody (catalog# sc-52, Santa Cruz Biotech, Dallas, Texas, USA) diluted 1:2000 in blocking solution; and then incubated for 2 h with Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Eugene, OR, USA) diluted 1:600 in blocking solution. The sections were washed with (PBS) after each incubation, and were finally mounted on glass slides using Aqua-Poly/Mount (Polysciences, Eppelheim, Germany).

For PV/cFos or SOM/cFos immunolabeling, amygdala sections were processed as described above for cFos immunohistochemistry. All secondary antibodies were diluted 1:600 in respective blocking solution and obtained from Invitrogen, Eugene, USA. For PV immunolabeling, sections were incubated for 12 h with mouse monoclonal anti-PV antibody (catalog# 235, SWANT, Bellinzona, Switzerland) diluted 1:2000 in blocking solution; and then incubated for two hours with Alexa Fluor 555 goat anti-mouse antibody. For SOM immunolabeling, the blocking solution contained 10% donkey serum and 0.3% Triton X-100 in PBS 0.1 M. Sections were incubated for 12 h in goat polyclonal anti-SOM antibody (catalog# sc-7819, Santa Cruz Biotech, Dallas, Texas, USA) diluted 1:1000 in blocking solution; and then with Alexa Fluor 488 donkey anti-goat antibody. Sections were washed with PBS after each incubation, and were finally mounted on glass slides using Aqua-Poly/Mount (Polysciences, Eppelheim, Germany).

Images of cFos and cellular markers were obtained using a confocal laser scanning microscope (Leica SP2) with a ×40 oil immersion objective. For each set, sections were anatomically matched and the settings for laser power, gain and offset were kept constant during imaging. 2 stacks of 5 μm thickness and containing 2 optical sections each were obtained from each amygdala section. In total, 10–12 stacks were imaged from 5–6 amygdala sections per mouse in each group. cFos images were thresholded manually in ImageJ with threshold set as 3*background intensity and the same threshold value was applied for WT, Nlgn2 KO, IgSF9b KO, and double KO in each group. Single-labeled cells were quantified using the Create Spots tool in Imaris (Bitplane, Zurich, Switzerland). To obtain the number of double- labeled cells, the Colocalize Spots tool in Imaris was used.

Retrograde labeling

Mice received an intraperitoneal (i.p.) injection of Carprofen (5 mg/kg) to reduce post-surgery pain 30 min prior to surgery. Mice were anaesthetized with Avertin by i.p. injection (20 ml/kg body weight) and placed in digital stereotaxic frame. To bilaterally label BA neurons projecting to the CeM, 50 nl of red Retrobeads (excitation maximum at 530 nm and emission maximum at 590 nm) were injected into the CeM (0.70 mm posterior, ± 2.35 mm medial, and 5.08 mm ventral from bregma). A Hamilton syringe (1 μl) was used to manually deliver the Retrobeads at the rate of 0.5 nl/sec. After the injection was completed, the tip of the syringe was raised by 100 μm and left for 3 min to allow diffusion of the Retrobeads at the injection site; and then slowly withdrawn at the rate of 1 mm/min. Following surgery, mice received Metamizol with drinking water (200 mg/kg/day, drinking rate estimated at 3 ml/day) for 3 days to reduce pain and risk of inflammation.

Mice were single housed for 7 days post-surgery to allow their full recovery and traveling of beads from the injection site up the axons of BA neurons to their somata. To induce anxiety-associated neuronal activation, mice were subjected to the OF for 10 min, and cFos was subsequently assessed as described above. Only slices from brains in which injection sites did not exceed the borders of CeM (confirmed by visualizing the sites of injection on 5 subsequent coronal sections of amygdala spanning 400 µm of tissue) were included in subsequent imaging and analysis. Data acquisition and quantification of cFos-positive cell bodies containing Retrobeads were performed similarly to quantification of double labeled neurons as described above. Sections were also used for histological verification of the Retrobead injection site, and mice were excluded from analysis if the injection site lay outside the CeM (Supplementary Fig. 3j).

In vivo electrophysiology

Mice were anesthetized with Avertin (loading dose 20 ml/kg, maintenance dose 2 ml/kg i.p.) and placed in a stereotaxic frame, and their body temperature was monitored by a rectal probe and maintained at 36 °C. An incision in the midline of the scalp was made to expose the skull. Bregma and lambda were aligned to a plane level ± 50 μm. A multi-wire electrode array was unilaterally implanted targeting the left CeM (0.9 mm posterior, 2.3 mm lateral and 5.04 mm ventral to bregma). The electrodes consisted of 2 bundles (spaced 750–950 μm) of 8 individual insulated tungsten wires (13 µm inner diameter, impedance 60–100 kΩ) inserted into a polymide tube (127 µm inner diameter) and attached to an 18-pin connector. A reference screw was implanted above the cerebellum. The implant was secured with two screws implanted in the skull ~300 µm lateral and anterior to the electrode and bonded with dental cement. Immediately after the surgery, mice subcutaneously received an analgesic (Carprofen 5 mg/kg) and an antibiotic (Baytril 5 mg/kg). Twenty four hours after the surgery, mice received Carprofen (5 mg/kg) subcutaneously and Baytril in the drinking water (0.2 mg/ml). Mice were sacrificed following the OF recording for histological verification of the recording site (Supplementary Fig. 4). Mice in which the electrode was not implanted in the CeM were excluded from the analysis.

Male mice were single housed for 7 days after the surgery and before the recording. For data acquisition, the mice were connected to the electrophysiological equipment and placed in the OF chamber, where they were allowed to explore for 15 min. The electrophysiological signal was amplified and sent to the acquisition board. The raw signal was acquired at 32 kHz sampling rate, band pass filtered (0.1–9000 Hz), and stored for offline analysis. During the experiment, simultaneous electrophysiological and video recordings were made by the Cheetah Data Acquisition System.

LFPs were analyzed using custom-written MATLAB scripts. The signal was filtered between 0.7 and 400 Hz using a zero-phase distortion FIR filter and down sampled to 1 kHz. The multitaper method was used for the power analysis (Chronux Package)52. The following time windows were used: theta range (4–12 Hz), 1 s with 0.8 s of overlap; beta range (18–30 Hz), 1 s with 0.5 s of overlap; gamma range, 0.15 s with 0.1 s of overlap. To calculate the power spectra during the entire OF session, 5 tapers were used with a time bandwidth of 3. For each mouse, the time periods and the tracks of the movement in the periphery and center were extracted using a modified version of the autotyping toolbox53. To evaluate the relative increase in beta power as a function of distance from the center, the distance from the center during the entire OF session was binned for each mouse (3 cm bins). To compute the beta power, the power was first summed across the beta band, and then the summed values were averaged for each location bin. The individual bin values were normalized by the average power in the periphery ( > 18 cm from center) and the linear correlation coefficients (fitlm function, MATLAB) per genotype were computed. To calculate the correlation between speed and beta power, the beta power was averaged in speed bins of 5 cm/s for each mice and the linear correlation coefficients computed as above.

To evaluate power changes during the SAP, the time events at which the mouse showed a clear SAP from the periphery towards the center of the OF were manually extracted. The events were identified by a typical elongation of the body and a very slow forward movement that was always followed by a retreat movement30,31. For the beta band, the power across frequencies was summed to produce one value for each time point. Then, the power values at each time point during the SAP were averaged and the mean power values per event were averaged per group. The Morlet wavelet transform was used to visualize the power at different frequencies ranges as shown in Fig. 3k, with 40 wavelets at centered frequencies ranging from 1 to 120 Hz and a length of 10 cycles.

Analysis of synaptic markers

Immunolabeling for VIAAT was performed on perfusion-fixed brains as described for the cFos assay. Briefly, sections were incubated for 24 h with rabbit polyclonal anti-VIAAT antibody (catalog# 131002, Synaptic Systems, Goettingen, Germany) diluted 1:1000 in blocking solution (3% bovine serum albumin, 10% goat serum, 3% Triton X-100 in 0.1 M PBS), washed, and then incubated for 2 h with Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Eugene, OR, USA) diluted 1:600 in blocking buffer. Immunolabeling for IgSF9b, Nlgn2, gephyrin, S-SCAM, GABA A Rα1, and GABA A Rγ2 was performed on methanol-fixed fresh frozen brain sections using a modified version of a published protocol54. Brains were frozen immediately after dissection in an isopentane bath at −35 °C to −40 °C. Coronal sections were prepared using a Leica CM3050S cryostat (Leica, Wetzlar, Germany), mounted on glass slides, and dried at room temperature. Sections were then fixed in methanol at −20 °C for 5 min, and blocked for 1 h. The duration of incubation was 12 h with primary antibody and 2 h with secondary antibody for each immunolabeling. The following primary antibodies were used, diluted in blocking solution: Rabbit polyclonal anti-IgSF9b (catalog# HPA010802, Sigma Aldrich, Darmstadt, Germany) at 1:1000 mouse monoclonal anti-Nlgn2 (catalog# 129511, Synaptic Systems, Goettingen, Germany) at 1:1000; mouse monoclonal anti-gephyrin (catalog# 147111BT, Synaptic Systems, Goettingen, Germany) at 1:1000; rabbit polyclonal anti-MAGI2 ( = S-SCAM, catalog# M2441, Sigma Aldrich, Darmstadt, Germany); rabbit polyclonal anti-GABA A Rα1 (catalog# 224203, Synaptic Systems, Goettingen, Germany) at 1:1000; guinea pig polyclonal GABA A Rγ2 (generously provided by Dr. Jean-Marc Fritschy, University of Zürich) at 1:1000. The following secondary antibodies were obtained from Invitrogen, Eugene, USA, and diluted 1:600 in the blocking solution: Alexa Fluor 555 goat anti-rabbit antibody, Alexa Fluor 488 goat anti-rabbit antibody, Alexa Fluor 555 goat anti-mouse antibody. Sections were washed with PBS after each incubation. The slides were then dried overnight at 4 °C, and covered with mounting media (Aqua-Poly/Mount; Polysciences, Eppelheim, Germany) and glass coverslips.

Images of synaptic markers were obtained using a confocal laser scanning microscope (Leica SP2) with a ×63 oil immersion objective and ×8 digital zoom. For each set, sections were anatomically matched and the settings for laser power, gain and offset were kept constant during imaging. 12 stacks of 2 μm thickness and containing 2 optical sections each were obtained from each amygdala section (12 stacks from 4 sections for each mouse in total). Images were thresholded in ImageJ, with same threshold applied to all mice in each set. To quantify perisomatic synapses, the perisomatic area was identified by manually tracing the perimeter of the cell body (defined as a circular area largely devoid of immunofluorescence)6,8. The perimeter was then expanded by 1.4 μm or 2 μm in each direction for quantification of postsynaptic puncta or presynaptic puncta, respectively. The number of particles was quantified in this area using the “count particles” module in ImageJ, and the number of particles per area was divided by the length of the cell body perimeter to obtain the final result.

In vitro electrophysiology

Adult (8–12-week-old) WT, Nlgn2 KO, IgSF9b KO, and double KO mice additionally expressing a VIAAT-Venus transgene32 were anesthetized with Avertin and perfused transcardially for 90 s with an ice-cold sucrose-based solution (6 mM MgCl 2, 0.1 mM CaCl 2, 50 mM sucrose, 2.5 mM glucose, and 3 mM kynurenic acid diluted in artificial cerebrospinal fluid (aCSF, 124 mM NaCl, 2.7 mM KCl, 26 mM NaHCO 3 , and 1.25 mM NaH 2 PO 4 )) as described previously for the preparation of amygdala slices from adult mice55. The brains were rapidly dissected and placed in the same ice-cold sucrose-based solution. The brainstem was removed and the brains were mounted on a holder and transferred to the vibratome chamber for preparation of 300 μm coronal sections. Slices containing the BA and central amygdala were transferred to a chamber filled with aCSF (see above) with additional 2 mM CaCl 2 and 1.3 mM MgCl 2 and equilibrated with 95% O 2 /5% CO 2 . Slices were allowed to recover for 20 min at 33 °C and maintained at room temperature thereafter. All chemicals were obtained from Merck Millipore (Molsheim, France), Sigma Aldrich (Darmstadt, Germany), or Tocris Bioscience (Bristol, UK).

Whole-cell patch-clamp recordings were obtained at room temperature (~22 °C) with an EPC10 amplifier (HEKA Elektronik, Germany). Slices were kept in a recording chamber and perfused with aCSF with additional 1.3 mM MgCl 2 , 2 mM CaCl 2, 18.6 mM glucose, and 2.25 mM ascorbic acid (osmolarity ≈320 mOsm) at a rate of 1–2 ml/min. Neurons were visually identified with infrared video microscopy using an upright microscope equipped with a ×60 objective. VIAAT-positive neurons were identified by Venus expression. For recordings in BA, VIAAT-Venus-negative neurons were targeted, while for recordings in CeM, VIAAT-Venus-positive neurons were targeted. Patch electrodes (3–5 MΩ open tip resistance when filled with internal solution) were pulled from borosilicate glass tubes. For voltage-clamp experiments to record miniature inhibitory postsynaptic currents (mIPSCs), patch electrodes were filled with Cs-based internal solution containing (in mM) 110 CsCl, 30 K-gluconate, 1.1 EGTA, 10 HEPES, 0.1 CaCl 2 , 4 Mg-ATP, 0.3 Na-GTP, and 4 N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314; Tocris-Cookson, Ellisville, MO); pH = 7.3 (adjusted with CsOH, 280 mOsm). To block glutamatergic EPSCs, 2 μM NBQX (6-cyano-7-nitroquinoxaline-2,3-dione) and 2 μM CPP ((RS)−3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid),) were added to the bath. Action potential (AP) firing was suppressed by adding 1 µM tetrodotoxin (TTX) to the aCSF. For current-clamp experiments and for recordings of spontaneous postsynaptic excitatory currents (sEPSCs), patch electrodes were filled with K-gluconate-based internal solution containing (in mM) 125 K-gluconate, 20 KCl, 0.2 EGTA, 2 MgCl 2 , 10 HEPES, and 2 Na 2 ATP; pH = 7.3 (adjusted with KCl, 280 mOsm). To block GABAergic EPSCs, 25 μM bicuculline methiodide (Tocris-Cookson, Ellisville, MO) was added to the bath. Firing thresholds were estimated from AP phase-plane plots and corrected for a measured liquid junction potential of 7.9 mV. To monitor series resistance on-line and to allow offline estimation of whole-cell membrane resistance and membrane capacitance, a voltage step (10 mV amplitude, 20 msec duration) was delivered at the beginning of each sweep during whole-cell voltage-clamp experiments. Capacitive current transients were analyzed using a simplified two-compartment equivalent circuit model56. Mean current transients obtained by averaging ≥ 30 consecutive sweeps were fitted using a double-exponential function \(I(t) = A_1 \times e^{ - t/\tau _1} + A_2 \times e^{ - t/\tau _2} + A_\infty\), where I(t) is the amplitude of the current at time t, A l and τ 1 denote the amplitude and time constant of the fast component of decay, A 2 and τ 2 represent the amplitude and time constant of the slower component of decay, and A ∞ , is the difference between the holding current and the final steady-state current at the end of the depolarizing pulse. The holding potential for whole-cell voltage-clamp recordings was set to −70 mV. Whole-cell voltage-clamp recordings were included in the analysis if the access resistance was initially ≤ 13 MΩ and did not change by more than 20% during the recording. Recordings with a leak current > 200 pA were rejected. Data were acquired with Patchmaster software (HEKA Elektronik, Germany), low-pass filtered at cut-off frequency of 5 kHz using a Bessel filter and digitized at 20 kHz. All offline analysis was performed with IgorPro (Wavemetrics, USA). mIPSCs were detected using a sliding template-matching algorithm implemented in IgorPro57.

Generation and stereotaxic injection of AAV

To generate shRNA-expressing AAV particles, IgSF9b and control shRNA sequences were first cloned into an AAV-shRNA-GFP vector58 generously provided by Dr. Ralph DiLeone (Yale University). The following shRNA sequences were used (modified from ref. 14): IgSF9b, TCATCAAGTTTGGCTACTAT; control (point mutant lacking knockdown activity), TCAT A AAGTT C GGCTACTAT. To confirm efficacy of knockdown, the resulting plasmids were co-transfected into HEK cells with a Myc-IgSF9b construct14 generously provided by Dr. Eunjoon Kim (Korea Advanced Institute of Science and Technology), and IgSF9b levels were quantified using standard immunoblotting procedures6 (primary antibody: rabbit anti-IgSF9b, Sigma, diluted 1:1000). AAV particles were generated using the pDPrs1/pDPrs2 packaging system (PlasmidFactory, Bielefeld, Germany) and the AAV-shRNA-GFP vectors described above. Plasmids were transfected into HEK cells using calcium phosphate transfection, and virus particles were harvested 72 h later58,59. To this end, HEK cells were lysed for 30 min at 37 °C in 20 mM Tris, pH 8.0, containing 150 mM NaCl, 0.5% sodium deoxycholate and benzonase, followed by incubation in 1 M NaCl at 56 °C for 30 min. Lysates were stored at −80 °C overnight, thawed, and purified on a 15%/25%/40%/54% iodixanol gradient by ultracentrifugation (90 min at 370,000× g). The 40% fraction was collected, diluted in PBS containing 1 mM MgCl 2 and 2.5 mM KCl, and concentrated using an Amicon 100 K MWCO filter.

Surgical procedures were exactly as described for the injection of Retrobeads, except that 1 μl of virus was injected bilaterally into CeA using a Nanoject II Microinjector (Drummond, Broomall, PA, USA) and a Micro pump controller (WPI). Mice were alternately assigned to receive AAV-control shRNA or AAV-IgSF9b-shRNA injections based on order of birth. The following coordinates relative to Bregma were used: AP (anteroposterior) −0.58, ML (mediolateral) ±2.48, DV (dorsoventral) and −5.4. After surgery, mice were housed in pairs and were allowed to recover for 6 weeks before assessment of behavior in the OF as described above. Mice were sacrificed following OF exposure for verification of the injection site as defined by GFP expression (Supplementary Fig. 5). Only mice in which both bilateral injection sites were correctly positioned in CeM were included in the study. Moreover, mice with any GFP expression in BLA or CeL were excluded, although minor, low-expression leakage into other border areas was tolerated. In total, 11 mice were excluded due to mistargeting (WT + Ctrl shRNA, 4 animals; WT + IgSF9b shRNA, 1 animal; Nlgn2 KO + Ctrl shRNA, 3 animals; and Nlgn2 KO + IgSF9b shRNA, 3 animals).

Statistical Analysis

Sample sizes were estimated based on prior experience with the methods used in this study6,8,60. All data were analyzed statistically using Prism (GraphPad Software, La Jolla, CA, USA) or Matlab. Outliers were identified using the Grubb’s test and were removed prior to statistical analysis. Behavioral scores were subjected to two-way ANOVA with post-hoc Tukey’s tests for comparison between groups. Data obtained from histological experiments were analyzed using two-way ANOVA with post-hoc paired, two tailed Student’s t-tests for comparison between groups. Data obtained from in vitro electrophysiological experiments were analyzed using two-way ANOVA with post-hoc Tukey’s test for comparison between groups. Distributions of mean mIPSC frequencies and amplitudes were analyzed using the Kolmogorov–Smirnov test. Data obtained from in vivo electrophysiological experiments were analyzed using two-way ANOVA with post-hoc Tukey’s test for comparisons between groups.

Code availability

Custom MATLAB scripts written for the analysis of LFPs are available from the corresponding author upon request.