Mice

All mice were group-housed and experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committees at the Massachusetts General Hospital and the Tufts University School of Medicine and NIH guidelines. All mice were housed in a 12-h (7:00 to 19:00) light–dark colony room at 22–24 °C, 30–50% humidity and with ad libitum access to food and water. Cntnap2^−/−, Pvalb^cre, Amigo2^cre and Rpl22^HA mice were obtained from the Jackson Laboratory (strain 017482, 017320, 030215 and 011029, respectively).

Viruses and virus constructs

AAV-S5E2-dTom-nlsdTom plasmid (plasmid 135630), AAV5-CamKIIa-hChR2(H134R)-eYFP (26969), AAV-S5E2-ChR2-mCherry (135634) and AAV PHP.eB virus were purchased from Addgene. AAV-S5E2-XPGs-nlsdTom viruses was generated by subcloning mouse Meis2 (NM_001346036.1), Bcl11a (NM_016707.3), Tbr1 (NM_009322.3) or dSACas9/VP64 into AAV-S5E2-dTom-nlsdTom plasmid. AAV-U6-sagRNA#1#2-Syn1-P2A-mCherry and AAV-Herc1gRNA#1-CAGGAAAAAGCCTGGTCTTCA-#2-AAAACAAATTCATGTGTATGT-Syn1-P2A-mCherry were generated by VectorBuilder. AAV-EF1a-DIO-mCherry virus was purchased from UNC. AAV-EF1a-DIO-Meis2-mCherry virus was generated by VectorBuilder. The Cal-Light plasmids pAAV-pCMV-Myc-TM-KA2-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA, pAAV-hSYN-M13-TEV-C-P2A-tdTomato and pAAV-TRE-eGFP were gifts from the Hyun Laboratory. AAV.PHP.eB viruses were produced by Boston Children’s Hospital Viral Core. Note, because of differences in constructs, S5E2-dTom-P2A-nlsdTom (control) results in dTomato distributed throughout the neuron, whereas S5E2-Meis2-P2A-nlsdTom is localized to the nucleus.

Immunohistochemistry

Mice were injected with viruses 2 weeks before perfusion. Mice were anaesthetized with ketamine and xylazine (10 mg ml^–1 and 1.6 mg ml^–1, respectively, intraperitoneally) and transcardially perfused with 4% paraformaldehyde (PFA), and their brains were removed and incubated in 4% PFA at 4 °C overnight. Brains were placed in 30% sucrose–PBS for 2 days and then embedded in medium (OCT, Fisher HealthCare). Cryosections (35 μm) were obtained (Leica) and stored in PBS (0.01% sodium azide) at 4 °C. For immunostaining, floating sections were permeabilized, blocked in blocking solution for 2 h (PBS containing 0.3 % Triton X-100 and 10% normal donkey serum) and then incubated with primary antibodies (PBS containing 10% normal donkey serum and 0.1% Triton X-100) at 4 °C overnight. Sections were then washed with PBS 3 times, 10 min each, then incubated with secondary antibodies in PBS for 2 h at room temperature. Sections were then washed with PBS 3 times, 10 min each, mounted on glass slides and coverslipped with DAPI Fluoromount-G (SouthernBiotech).

Image analysis

For PV puncta and SYT2 puncta, images were obtained from three sections per mouse hippocampus blind to treatment and genotype. A Leica SP8 confocal laser microscope and LAS software were used to capture images in the stratum lucidum at high-resolution (2,048 × 2,048 pixels). Single confocal-plane images were captured in the CA2 and CA3ab subfields using a ×63 oil objective plus ×43 digital zoom. For quantification of sample sizes of PV⁺ puncta and SYT2⁺ puncta, densities were averaged from 18 images per mouse for CA3 and 12 images per mouse for CA2. Puncta were analysed using the StarDist 2D plugin and particle analysis tools in Fiji ImageJ. Threshold values were held constant across images. For Cal-Light, dCA3a cells were manually counted. To reduce the contribution of background fluorescence, a threshold based on maximizing Yen entropy⁶² was applied to GFP images during quantification.

Antibodies

The following antibodies were used in the study: PV (rabbit, Swant PV25, 1:5,000, RRID: AB_10000344; goat, Swant PVG213, RRID: AB_2721207, 1:1,000); RGS14 (mouse, NeuroMab 75-170, RRID: AB_2179931, 1:500; rabbit, Proteintech 16258-1-AP, RRID: AB_2179918, 1:500); SYT2 (mouse, Abcam AB154035-1001, RRID: AB_2916272, 1:250); RFP (rabbit, Rockland 600-401-370, RRID: AB_2209751, 1:1,000; goat, Sicgen AB1140-100, RRID: AB_2877097, 1:500); FOS (guinea pig, Synaptic Systems 226-004, RRID: AB_2619946, 1:3,000); GFP (chicken, Invitrogen A10262, RRID: AB_2534023, 1:500); gephyrin (rabbit, Synaptic Systems 147-008, RRID: AB_2619834, 1:500); MEIS2 (rabbit, Protein Tech 11550-1-AP, RRID: AB_2143028, 1:400); BCL11A (mouse, Abcam ab19487, RRID: AB_444947, 1:500); TBR1 (rabbit, Abcam ab31940, RRID: AB_2200219, 1:200); GFAP (Chicken, Millipore AB5541, RRID:AB_177521, 1:2,000); IBA1 (rabbit, FujiFilm 019-19741, RRID: AB_839504, 1:500); and SST (mouse, Santa Cruz G-10, RRID: AB_831726, 1:500). The following fluorescent-label-coupled secondary antibodies from Jackson Immuno Research were used at 1:500 dilution: Alexa-Fluor-488-conjugated donkey anti-rabbit IgG (711-545-152); Cy3-conjugated donkey anti-rabbit-IgG (711-165-152); Alexa-Fluor-488-conjugated donkey anti-mouse-IgG (715-545-151); Cy3-conjugated donkey anti-mouse-IgG (715-165-151); Alexa-Fluor-488-conjugated donkey anti-chicken-IgG (703-545-155); Cy3-conjugated donkey anti-goat-IgG (705-165-147); 647-conjugated donkey anti-guinea pig-IgG (706-605-1481); and Cy3-conjugated donkey anti-guinea-pig-IgG (706-165-148).

RNA in situ hybridization and immunofluorescence imaging

RNAscope in situ hybridization was performed following the RNAscope Multiplex Fluorescent Reagent kit v.2 protocol (323100, Advanced Cell Diagnostics). In brief, mice were perfused with PBS followed by 4% PFA. Brain slices (10 µm) were mounted and dried at –20 °C for 2 h. The slides were washed in PBS, then dehydrated in a gradient of ethanol (50%, 70%, 100% and 100%) for 5 min each and air-dried for 5 min. RNAscope hydrogen peroxide was applied to tissues and incubated at room temperature for 10 min, followed by treatment with RNAscope 1× Target Retrieval Reagent. Tissues were incubated for 10 min at 95 °C followed by washes in ddH₂O and 100% ethanol. Tissues were dried at room temperature for 3 min. RNAscope Protease III was applied and incubated for 30 min at 40 °C, then washed with water. The target probes were prepared (MmRgs14, 416651; Mm-Pvalb-C2, 421931-C3; and Mm-Meis2-C3, 436371-C3). Target probes were applied and incubated at 40 °C for 2 h, after which tissues were washed with 1× wash buffer and placed in 5× SSC buffer overnight. Tissues were washed with wash buffer twice followed by incubation with AMP1, AMP2 and AMP3 (30 min for AMP1 and AMP2, 15 min for AMP3, at 40 °C). Tissues were washed with wash buffer twice in between each amplification step, then incubated with HRP specific to each channel (15 min, 40 °C). Tissues were washed twice, after which the TSA fluorophore specific to each channel or probe (Opal 520, 570 and 690, Akoya Biosciences) was added and incubated for 30 min at 40 °C. Tissues were washed twice and incubated with RNAscope HRP Blocker for 15 min at 40 °C. Fluorescent images were captured using a SP8 Leica confocal microscope. All Pvalb⁺ cells were outlined and Meis2 intensity was quantified.

RNA sequencing

Tissue collection and RNA isolation

Hippocampal CA2/CA3 regions were collected following injections of lentivirus expressing shAblim3 or shNT into the DG of 2-month-old PV^cre;Rpl22HA^f/f mice for 2 weeks. Brain tissues were snap-frozen and pooled from 6 mice (male and female) for every sample. Samples were immunoprecipitated with anti-HA magnetic beads (Pierce, 88836) for 3 h at 4 °C^30,63. After elution using a RNeasy Plus Micro kit (Qiagen), purified ribosome-associated RNAs were stored at –80 °C. RNA quality was assessed using a TapeStation (Agilent), and RNA amounts were quantified using a Qubit 4.0 Fluorometer (Life Technologies). Only RNA samples with a RIN value above 8.0 were used for library preparation and sequencing.

Library preparation, Illumina sequencing and bioinformatics

NGS libraries were constructed from total RNA using a Clontech SMARTer v.4 kit (Takara), followed by sequencing on an Illumina HiSeq 2500 instrument, which resulted in 20–30 million 50 bp reads per sample. STAR aligner⁶⁴ was used to map sequencing reads to the transcriptome in the mouse mm9 (GRCm37) reference genome. Read counts for individual genes were produced using the unstranded count function in HTSeq (v.0.6.0)⁶⁵, followed by the estimation of expression values and detection of differentially expressed transcripts using EdgeR⁶⁶. Only the genes with count per million reads of 1 for one or more samples⁶⁷ were included for subsequent analyses. DEGs were defined by at least 1.5-fold change with FDR < 0.05.

RT–qPCR analysis

Hippocampal DG and CA2/CA3 regions were collected and snap-frozen⁶⁸. In brief, cDNA samples were reverse transcribed from RNA collected from PV^cre;Rpl22^HA/HA mice injected with either shNT or shAblim3 into DG. Total RNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific), and then equal amounts of RNA were used for reverse transcription (SuperScript IV First-strand synthesis system, Invitrogen). RT–qPCR was carried out using SYBR green (Bio-Rad) and primers (Primer bank)⁶⁹ with the following sequences: Ablim3-F 5′-GGTCCGTGTCCACAACAAC-3′; Ablim3-R 5′-GTCCCGGCAGCTATCACAG-3′; Bok-F 5′-CCACAGACAAGGAGCTGGT-3′; Bok-R 5′-TAGCCAAGGTCTTGCGTACA-3′; Dsp-F 5′-CGGACATTCATGCGAGATAC-3′; Dsp-R 5′-GCCTTGAACTGGGAACACTC-3′; Bcl11a-F 5′-TGGTATCCCTTCAGGACTAGGT-3′; Bcl11a-R 5′-TCCAAGTGATGTCTCGGTGGT-3′; Meis2-F 5′-CAGGGTGGTCCAATGGGAATG-3′; Meis2-R 5′-GGGGGTCCATGTCTTAACTGAG-3′; Tbr1-F 5′-CAAGGGAGCATCAAACAACA-3′; Tbr1-R 5′-GTCCTCTGTGCCATCCTCAT-3′; Herc1-F 5′-GAAGATGTGGATGCAGCAGA-3′; Herc1-R 5′-GGTCTGTCCGGTGAAGGATA-3′; Gapdh-F 5′-GCTTGTCATCAACGGGAAG-3′; Gapdh-R 5′-TTGTCATATTTCTCGTGGTTCA-3′. Primers for the following MEIS2 downstream candidates were used: Cdh2-F 5′-AGCGCAGTCTTACCGAAGG-3′; Cdh2-R 5′-TCGCTGCTTTCATACTGAACTTT-3′; Chd7-F 5′-GGAGAACCCTGAGTTTGCTAGCGCAGTCTTACCGAAGG-3′; Chd7-R 5′-CCCTGAAGTAGAGGCGACAG-3′; Kirrel3-F 5′-TCGGAGAATGAATGAAGGTCAAG-3′; Kirrel3-R 5′-GATGGCACACAGCAGAGTCA-3′; Reln-F 5′-CTGTGTCATACGCCAAGAACA-3′; Reln-R 5′-GGGGAGGTACAGGATGTGGAT-3′; Syt2-F 5′- AGAACCTGGGCAAATTGCAGT-3′; Syt2-R 5′-CCTAACTCCTGGTATGGCACC-3′; Tanc1-F 5′-CGAGCGCCACACACATAAC-3′; Tanc1-R 5′-AGGCAGTGCTGCTTGTGAG-3′; Kcnab1-F 5′-TTCCGCACTGTCGCTATCATC-3′; Kcnab1-R 5′-AGCATGAAACTCTGAGTCCTGA-3′; Pvalb-F 5′-CATTGAGGAGGATGAGCTG-3′; and Pvalb-R 5′-AGTGGAGAATTCTTCAACCC-3′.

Gene set enrichment analysis

Gene set enrichment analysis was performed using the package from a joint project of UC San Diego and the Broad Institute (https://www.gsea-msigdb.org/gsea/index.jsp), using FDR < 0.05 and default settings. Mouse MSigDB (v.2024.1.Mm) was used for pathway (Gene Ontology terms) analysis^70,71.

Stereotactic viral injection

Mice were administered carprofen (5 mg kg^–1, subcutaneously) before surgery and were then anaesthetized with ketamine and xylazine (10 mg ml^–1 and 1.6 mg ml^–1, intraperitoneally) or with 1.75% isoflurane (Baxter Healthcare Med Delivery Systems). Mice were placed in a stereotaxic frame, and a small hole was drilled at each injection site (Foredom K.1070 High Speed Rotary Micromotor kit). Bilateral injections were performed using Hamilton microsyringes (Hamilton, Neuros Syringe 7001) or Nanoject digital microinjectors that were slowly lowered into target sites and remained in place for 8 min before viral infusion at a rate of 50 nl min^–1. The following coordinates relative to bregma were used: dorsal DG: –1.8 mm (anterior–posterior (AP)), ±1.35 mm (medial–lateral (ML)) and –2.25 mm (dorsal–ventral (DV)); dorsal CA2/CA3: –1.8 mm (AP), ±2.45 mm (ML) and –2.35 mm (DV). Recombinant AAVs (titre of 1 × 10¹³) were injected for a total volume of 100 nl per injection site. After 10 min of infusion, the microsyringes were slowly withdrawn and the skin above the incision was sutured with coated vicryl sutures. For Cal-Light tagging experiments⁴⁵, viruses were mixed (1:1:1 ratio) and injected in a total volume of 300 nl. One week later, animals were injected with AAV-S5E2 control virus or AAV-S5E2-Meis2 virus and implanted with an optical fibre probe above CA2/3 and given 2 weeks to recover before behavioural testing. The following coordinates relative to bregma were used: –1.80 (AP), ±2.28 (ML) and –2.20 (DV) for virus injection; –1.80 (AP), 2.28 (ML) and –1.80 (DV) for optical fibre probe implantation. After surgery, mice were placed in a clean empty cage on top of a heating pad with ambient temperature to 36 °C until full recovery from anaesthesia. Mice were monitored and received a daily injection of carprofen (5 mg kg^–1, intraperitoneally) for 3 days after surgery⁹.

Electrode implantation

Mice were allowed to recover and 2 weeks later underwent a second surgery of tetrode implantation. Mice were placed in a stereotaxic frame after anaesthesia (1.75% isoflurane) to implant three bone screws and a 16-channel, custom-built, twisted wire electrode bundle. A four-wire electrode bundle was created by twisting together four 75-µm diameter nichrome wires (California Fine Wire). The bundle was cut at an angle that spanned 0.5 mm. Four different sites were chosen for twisted wire implantation: the tips of the twisted wire were placed in the dorsal hippocampus at the following locations: DG: –1.8 mm (AP), +1.35 mm (ML) and –2.25 mm (DV); CA1, –1.8 mm (AP), +1.35 mm (ML) and –2.18 mm (DV); CA3, –1.8 mm (AP), +2.45 mm (ML) and –2.35 mm (DV); and CA2, –1.8 mm (AP), +2.45 mm (ML) and –2.31 mm (DV). The twisted electrodes were each attached to a pin in a Mill-Max connector. After confirming that the recording electrode array extended through CA1, the wires and connectors were secured in place with dental cement. The Omnetics connector of the recording electrode and the Mill-Max connector of the electrode bundle were anchored to the skull along with bone screws using dental cement mixture (C&B Metabond, Parkel, and TEETs Denture Material, Cooralite Dental Mfg).

Recording setup

The implanted mice were attached to the headstage (RHD 32 ch, C3314, Intan Technologies) through a custom-made Omnetics to Mill-Max adaptor (Mill-Max model 852-10-100-10-001000). Behaviour was recorded using a monochromatic camera (30 frames per s (fps), Flea3 USB3, FLIR). Electrophysiological signals were sampled at 30 kHz and recorded using an Open Ephys Acquisition system via an ultrathin SPI interface cable connected to the headstage board. Electrophysiological recordings were synchronized with recorded video using a TTL trigger pulse and by recording camera frame strobes. The implanted mice (10 weeks old) were allowed to explore their home cage for 2 h (pre-social), followed by 5 min of social interaction in the home cage with a novel, sex-matched juvenile mouse. After the social encounter, another 2 h of home-cage exploration and recording were conducted. Mouse movement was analysed on the basis of markerless tracking of the snout using DeepLabCut⁷². A snout speed of less than 1.5 cm s^–1 in the home cage was considered an immobility period.

Extracting NREM periods

LFP data processing and ripple analysis were conducted using a custom written Python script. Initially, the LFP data from the selected CA1 channel was scaled to microvolts and downsampled to 1,000 Hz. The NREM sleep periods were identified in the downsampled CA1 LFP by first computing a spectrogram (10-s window, 1-s step, 0–300 Hz). Features, including the first principal component (PC1, derived from the z-transformed 0–300 Hz spectral power) and theta dominance (5–10 Hz power/2–16 Hz power) were then extracted. NREM states were defined as periods in which PC1 exceeded its 75th percentile for at least 5 s, accompanied by a low theta dominance ratio⁷³.

Ripple analysis

Ripple events were subsequently detected in these NREM epochs from the downsampled CA1 LFP. The LFP was bandpass-filtered (100–250 Hz, third-order Butterworth) and z-scored. To calculate the power envelope of this ripple-band signal, the z-scored filtered signal was rectified and then filtering again, in the 1–20 Hz band, using third-order Butterworth^74,75. Ripple events were marked when this power envelope exceeded 2 Z, reached a peak greater than 5 Z, had a duration between 20 and 150 ms and were merged if the inter-event interval was less than 20 ms^76,77. An artefact rejection was performed when the z-scored envelope of the DG channel concurrently exceeded the high threshold, thereby coinciding with the detected ripple event in the CA1 channel. For each valid ripple event, the following properties were extracted: the event start time (the point at which the power envelope went above 2 Z); the end time; the peak power time (the point at which the power envelope fell back below 2 Z); and the peak LFP time (the point between the start and end times at which the highest amplitude ripple-band cycle occurs). These provided precise timestamps for the boundaries of the event and its peak activity points. The duration of each ripple was calculated in milliseconds, which represented its total temporal extent. The peak power quantified the maximum deviation of the power envelope of the ripple from the baseline, normalized by its median and median absolute deviation. The peak LFP amplitude was determined as the maximum absolute value the detected boundaries of each ripple. For visualization of each ripple, time–frequency power was calculated by applying a continuous complex Morlet wavelet transform to the raw LFP signal (0–300 Hz) surrounding the event using PyWavelets⁷⁸. Power was estimated for frequencies from 100 to 250 Hz, and the resulting spectrograms were generated on a decibel (dB) scale.

Behavioural procedures

Two weeks after viral injections, mice were handled for 3 days before behavioural experiments to habituate them to human handling and transportation from the vivarium to the behavioural testing rooms. The behavioural assays were performed in the following order: open-field (OF, day 1), visual cue habituation (day 2), novel object location (NOL) followed by novel object recognition (NOR, day 3), and social recognition and discrimination (day 4). All the behavioural assays were performed in the same chambers (40 × 40 cm, MazeEngineers). Videos were recorded and exported from Freezeframe (Actimetrics) and analysed using EthoVision XT 15 (Noldus). Centre point tracking was used to record movement, and nose point tracking was used to evaluate object and social interaction. An interaction was registered when the test mouse’s nose position to object or stimulus mouse was within 1 cm⁹.

Behavioural paradigm for PV IN synapse, SYT2 and puncta analysis

Mice were handled for 7 days before behavioural experiments to habituate them to human handling, transportation from the vivarium to the behavioural testing rooms and the context for 1 h. On day 8, mice were either exposed to context only or with one stimulus mouse in one pencil cup for 10 min. Mice were returned to their home cage and subsequently perfused 90 min later.

Behavioural paradigm for Meis2 and MEIS2 intensity analysis

Mice were handled for 3 days before behavioural experiments to habituate them to human handling, transportation from the vivarium to the behavioural testing rooms and the context for 1 h. On day 4, mice were either exposed to context only or with one stimulus mouse in one pencil cup for 10 min. Mice were returned to their home cage and subsequently perfused 10 min later.

OF paradigm

Mice were transported into a holding room and habituated for 1 h before testing. The total distance travelled and the time spent in the centre of the arena were quantified over 30 min⁹.

NOL and NOR

Two identical objects (2 × 4 × 6 cm) were placed in the OF chamber along one side (5 cm distance from the wall). Mice were placed in the opposite side of the object and allowed to explore freely for 5 min and then returned to their home cage for 2 h. Then one object was moved to the opposite side, and mice were placed in the middle of objects and allowed to explore for 5 min. Mice were returned to home cage for 10 min. Next, one object was replaced with a novel object (4 × 4 × 6 cm, 5 cm distance from the wall), and mice were placed in the middle of the objects and allowed to explore freely for 5 min. The time spent exploring the objects (noise point within 2 cm) was quantified⁹.

Social recognition and social discrimination

Stimulus mice (matched for strain, age and sex) were habituated to being placed in a pencil wire cup in the OF chamber before the task day for 3 days (15 min per day). The task consisted of three trials with 5 min of inter-trial intervals: habituation (empty cup versus empty cup); recognition (empty cup versus stimulus); and discrimination (novel versus familiar). Mice were placed in the centre of the chamber for 10 min. The locations of the familiar and novel stimuli were counterbalanced across trials. The time spent exploring the stimulus mouse was quantified (nose point within 1 cm)⁹.

Tagging socially active neurons with soma-targeted Cal-Light

Behavioural paradigm and tagging protocol

Before testing, animals were placed in holding cages for a minimum of 1 h, using the same cage each day. Subject mice underwent a 3-day habituation protocol, during which they explored an OF chamber with two empty cups placed in opposite corners while attached to lightless fibre optic cables on the final 2 days. One day after habituation, subject mice were exposed to a novel stimulus mouse (stimulus 1) placed under one of the cups and allowed to explore the chamber for 10 min. When subjects approached within 5 cm of the stimulus mouse, blue light was delivered through fibre optic cables to mediate Cal-Light tagging of the engram. Immediately after the session, subjects were returned to their holding cages. Four hours after initial exposure, subjects were reintroduced to the chamber for a second 10-min session, with a familiar mouse under the same cup as before. Following this, subjects were returned to holding cages and subsequently perfused 90 min later. For social versus contextual comparison of Cal-Light activity, no mouse was placed under the cup in the first second 10-min session. For light versus no light comparisons, mice underwent one 10-min social session with a novel mouse, with or without blue light delivery though fibre optic cables, and were perfused 5.5 h later.

Optical fibres and laser delivery

Optical fibres were constructed using a 200 μm core, 0.37 numerical aperture multimode fibre from Thorlabs. Fibres were inserted through a 230 μm core zirconia ferrule 701 (Precision Fiber Products) and glued in place before thoroughly polishing for a smooth connection between the fibre and optical cable from the laser. For Cal-Light activation, a 100 mW 475 nm blue laser diode was used (OEM Laser Systems). When the subject animal was within a predefined zone, the light was delivered at a frequency of 1 kHz and an intensity of 5–7 mW using an external arbitrary waveform generator (Agilent) attached to the signal port of the laser.

Ex vivo electrophysiology

Mice were 2–3 months old before viral transduction (see above for stereotactic coordinates). At 2–3 weeks after viral infusion, mice were anaesthetized with ketamine and xylazine (10 mg ml^–1 and 1.6 mg ml^–1, respectively, intraperitoneally) then transcardially perfused with ice-cold (4 °C) choline chloride-based artificial cerebrospinal fluid (ACSF) composed of (in mM) the following factors: 92 choline chloride, 2.5 KCl, 1.25 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose and 10 MgSO₄·7H₂O. Mouse brains were rapidly extracted following decapitation. Coronal slices (300-µm thick) containing the dorsal hippocampus were cut in ice-cold (4 °C) choline chloride ACSF using a Leica VT1000 vibratome (Leica Biosystems) and transferred to warm (33 °C) normal ACSF for 30 min. Normal ACSF contained (in mM) the following factors: 124 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 24 NaHCO₃, 5 HEPES, 12.5 glucose, 2 MgSO₄·7H₂O and 2 CaCl₂·2H₂O. All ACSF solutions were adjusted to pH 7.4 and mOsm of 305, and were continuously saturated with carbogen (95% O₂ and 5% CO₂). Slices were allowed to cool to room temperature (20–22 °C) for 1 h before recordings.

Whole-cell patch-clamp recordings were amplified, low-pass filtered at 1.8 kHz with a 4-pole Bessel filter and digitized (Muliclamp 700B, Digidata 1550B, Molecular Devices). Slices were placed in a polytetrafluoroethylene submersion chamber and continually perfused with normal ACSF (>2 ml min^–1). Neurons were visually identified by infrared differential interference contrast imaging combined with epifluorescence using LED illumination (pE-300 white, CoolLED). PNs in CA2 and CA3ab were distinguished by their anatomical location and distinct electrophysiological properties. Borosilicate patch pipettes had a resistance of 4–5 MΩ and filled with an internal solution containing (in mM) the following factors: 120 CsMeS, 4 MgCl₂, 1 EGTA, 10 HEPES, 5 QX-314, 0.4 Na₃GTP, 4 MgATP, 10 phosphocreatine and 2.6 biocytin, at pH 7.3, 290 mOsm. For current-clamp recordings, patch pipettes were filled with 130 mM potassium gluconate in place of CsMeS and QX-314 was excluded. Once the GΩ seal was obtained, neurons were held in voltage-clamp configuration at –70 mV and the input resistance, resting membrane potential and capacitance were measured. Series resistance (<30 MΩ) was monitored throughout recordings, and recordings were discarded if the series resistance changed by >20% from baseline.

Optically evoked EPSCs and IPSCs were evoked with 1 ms 473 light pulses delivered above the mossy fibre pathway–the hilus of the DG. Current responses were recorded at 1.5× threshold, defined as the minimum stimulation intensity required to produce a consistent current response beyond baseline noise. Isolation of EPSCs was done by voltage clamp at –70 mV and IPSCs at 0 mV. Optical 10-pulse stimulation trains were evoked 5 times at an interval of 20 s between trains. The inter-event interval between pulse stimulation was 100 ms.

Electrically evoked EPSCs and IPSCs were evoked with 0.2 ms stimulation from a bipolar tungsten electrode in a patch pipette with a resistance of 1–2 MΩ and filled with ACSF. The stimulation pipette was placed on the mossy fibre pathway–the hilus of the DG or the stratum oriens between CA2 and CA3a to elicit Schaffer collateral stimulation. Pharmacological validations were conducted to ensure selective mossy fibre stimulation or selective Schaffer collateral stimulation using DCG-IV (1 µM) or tetrodotoxin (TTX) and 4AP (1 µm and 100 µm, respectively).

LTP of the EPSCs and long-term depression of the IPSCs (iLTD) was induced by electrically evoked TBS, which consisted of bursts of 4 pulses at 100 Hz, with and inter-burst interval of 200 ms, and repeated 4 times at 10 s intervals. A bipolar tungsten electrode in a patch pipette was placed on the mossy fibre pathway. Stimulation was set to 1.5× threshold of the resulting EPSC. The recording consisted of a 10 min baseline of the optically evoked EPSC or IPSC, TBS, then 40 min of the optically evoked postsynaptic current. Plasticity was calculated on the basis of the per cent change from the last 5 min of the baseline current amplitude to the last 5 min of the 40 min recording.

A current-clamp configuration was used to record intrinsic membrane properties. To assess action potential spiking activity, the neuron was clamped at –70 mV and 10 pA ascending step currents were delivered at 500 ms durations. Bursting neurons were identified by their asynchronous rapid firing within the first 50 ms of the current step followed by failure to fire and were excluded from analyses. In some experiments, intrinsic membrane properties were recorded independent of excitatory synapses by bath application of cyanquixaline (CNQX, 10 µM), an AMPA and kainite receptor competitive antagonist, and d-2-amino-5-phosphonopentanoic acid (AP5, 50 µM), a selective NMDA receptor antagonist.

sEPSCs and sIPSCs were recorded by voltage clamp at –70 mV for sEPSCs and 0 mV for sIPSCs. Pharmacological validations were conducted to ensure no sIPSCs were visible during sEPSC recordings and vice versa (data not shown). No inward events were detected after bath application of CNQX (10 µM) while voltage clamped at –70 mV. Likewise, no events were detected after bath application of gabazine (10 µM), a GABA_A receptor antagonist, while voltage clamped at 0 mV.

mEPSCs and mIPSCs were recorded after bath application of TTX (1 µM). Isolation of mEPSCs was done by voltage clamp at –70 mV and mIPSCs at 0 mV. Autodetection parameters for the inclusion of spontaneous and miniature events were determined by calculating the minimum threshold: root mean square 2 × 1.5. Data acquisition was performed using Clampex and analysed with Clampfit (Molecular Devices v.11) and EasyElectrophysiology (v.2.8.0) software.

ECoG surgical implant

All animal surgeries and subsequent experiments were conducted in accordance with Tufts University’s Institutional Animal Care and Use Committee guidelines and animal use protocols. In brief, 6–7-month-old Cntnap2^−/− and Cntnap2^+/+ mice that had previously undergone CA2/CA3 AAV injection were anaesthetized with isoflurane (3% induction, 1.5% for maintenance, 2 l min^–1 O₂ flow rate) and given systemic analgesic (buprenorphine, 0.1 mg kg^–1, subcutaneously) as well as local analgesia near the incision site (bupivacaine, 4 mg kg^–1, subcutaneously). Mice were then placed in a stereotactic frame, and the dorsal side of the skull was shaved and disinfected with iodine and ethanol. A scalp incision was made, and the skull surface cleaned and dried. Four burr holes were drilled into the skull without puncturing the dura mater. The drill bit tip diameter was 0.7 mm (item no. 19007-07, Fine Science Tools). Stereotaxic coordinates of the four drill holes are as follows: anterior burr holes were −0.6 mm (bregma, AP axis) and 2 mm left (ECoG) and right (ground) of the midline (ML); posterior burr holes were – 2.6 mm (bregma, AP) and 2.5 mm left (reference) and right (ECoG) to ML. Four 0.25 cm stainless steel screw electrodes with attached silver wires (part no. 8403, Pinnacle Technologies) were gently screwed into the burr holes and fixed with dental cement and superglue to increase implant stability for chronic ECoG recordings. The silver wires of the electrode screws were then soldered to the headmount (part no. 8402, Pinnacle Technologies). The headmount–electrode construct was then secured to the skull with dental cement. After surgery, animals were allowed to recover for at least 7 days before chronic ECoG recordings started and were given analgesia (buprenorphine, 0.1 mg kg^–1, subcutaneously) as needed for the first 3 days after surgery.

Chronic ECoG recording

After recovering from surgery, a preamplifier (100× gain, 1 Hz high-pass filter, part no. 8213, Pinnacle Technologies) was plugged into the implanted headmount and preamplifiers were attached to a commutator in the ECoG recording system (Pinnacle Technologies). Mice were housed in a round, acrylic ECoG recording chamber with access to food and water and kept on a standard light–dark cycle. ECoG data were recorded using LabChart Pro software (AD instruments) with a 1 kHz sampling rate. ECoG data were collected continuously 24 h a day for at least 14 days. After recording, ECoG signals were filtered (100 Hz low-pass filter) and recordings were manually reviewed manual by an experienced, blinded investigator. When seizures were identified, their duration was quantified, and their time of incidence was noted.

SLEAP

Mice were pair-housed for over 2 weeks before recording. On the recording day, the cage lid was removed, and the cage was placed inside a recording chamber overnight with access to food and water. Behaviour was continuously recorded for 8 h under infrared lighting, with a 1 h segment, captured 1 h after the cage was placed in the chamber, selected for analysis. We used SLEAP⁴⁴ to track and estimate the poses of freely behaving mice in their home cages. To analyse social interactions between mice, a multi-instance, bottom-up model with a U-Net architecture was trained on 2,751 instances across 917 frames, with 306 validation instances from 102 frames. Six body parts were labelled as nodes: nose, right ear, left ear, head, body centre and tail base. Our average distance between the predicted and ground truth nodes was 4.3 pixels. Training frames were randomly selected from videos containing different mice, with slight variations in contrast between subjects and background. Recordings were captured continuously during a 1-h session, and the frame rate was 23.97 fps. A batch size of 6 was used for training, with data augmentation through rotation (360°).

For inference, identity tracking across frames was performed using a flow tracker, with centroid similarity and the Hungarian matching algorithm applied over an elapsed frame window of 4 frames. Instances were manually inspected for identity switches and corrected before data export. Data were analysed using a Python script modified from SLEAP analysis examples (https://sleap.ai/notebooks/Analysis_examples.html). These were used to analyse social interactions based on the distance between nodes. First, missing nodes were linearly interpolated, and predictions were smoothed using a Savitzky–Golay filter to reduce jitter. We then measured nose-to-nose, nose-to-centre and nose-to-rear distances. Interactions were defined using the following pixel thresholds: 61 pixels (nose-to-nose), 100 pixels (nose-to-centre) and 70 pixels (nose-to-rear). Thresholds were verified against manually scored data to ensure accuracy.

Sex as a biological variable

Male and female mice were included in most of the experiments in this study. Attempts were made to balance sex in each experimental group. However, we acknowledge that the ratio of males and females are not consistent across every group, and that group sizes may be underpowered for statistical comparisons of sex.

Statistics, rigour and reproducibility

All experimenters were blind to treatment conditions throughout data collection, scoring and analyses. Animals were assigned to experimental groups based on genotype. Representative images and traces were selected from three or more independent variables in each experiment (mice). Statistical analyses were conducted using Prism (v.10; GraphPad) and the minimum sample size was determined on the basis of previous experience of experimental paradigms, existing literature and power analyses. Significance was defined as P < 0.05 and exact P values are provided whenever possible. Appropriate nonparametric tests were used when datasets did not meet parametric assumptions. Grubbs’ or ROUT tests were used to identify outliers with α or Q = 0.05 (Fig. 4k). Detailed statistical analyses are provided in Supplementary Table 3.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.