Experimental models and subject details
Sprague–Dawley Nlgn3−/y transgenic rats created by Horizon Discovery, now Envigo (RRID: RGD_11568700) were housed on either a 14/10 h (Bangalore Biocluster) or 12/12 h (University of Edinburgh) light/dark cycle with a 21 ± 2 °C room temperature and food/water ad libitum. Animal husbandry was carried out by University of Edinburgh or Bangalore Biocluster technical staff. Rats were housed 4 per cage (2 WT, 2 Nlgn3−/y, littermates where possible) in conventional non-enriched cages, except for rats that had undergone surgeries, which were single-housed in individually ventilated cages. Body weight was monitored throughout experiments.
Experiments carried out in Edinburgh included: RNA sequencing and Western Blotting (Fig. 1), acute slice whole-cell electrophysiology recordings (Fig. 5, Additional file 1: Fig. S5), and in vivo electrophysiology and behaviour experiments (Figs. 6, 7, Additional file 1: Figs. S6–8).
Experiments carried out in Bangalore included: Western Blotting, auditory fear conditioning (Fig. 2), contextual fear conditioning (Additional file 1: Fig. S1), active place avoidance (Fig. 3, Additional file 1: Fig. S3), shock-ramp test (Fig. 4), open field (Additional file 1: Fig. S3A), marble interaction time (Additional file 1: Fig. S3E) and tail-flick test (Additional file 1: Fig. S4B).
Rats were handled for a minimum of 3 days prior to behavioural testing. Animals undergoing fear conditioning and active place avoidance tasks underwent marble burying, open field, object recognition memory tasks and three-chamber task prior to those shown in this study.
Male littermates were assigned to experimental groups based on genotype to achieve balanced cohorts. Genotyping was carried out by Transnetyx Inc. All experiments and analyses were performed blind to genotype.
P60-90 male WT and Nlgn3−/y rats were anaesthetised with gaseous halothane and decapitated. The brain was extracted and cooled in ice-cold (> 4 °C) carbogenated (bubbled with 95% O2/ 5% CO2) cutting artificial cerebrospinal fluid (cACSF, 87 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 25 mM glucose, 3.4 M sucrose, 7 mM MgCl2, 0.5 mM CaCl2) before slicing medial-prefrontal cortex. Slices were snap frozen on dry ice and stored at −80 °C.
RNA was isolated as previously described , and RNA integrity values determined using an Agilent 2100 Bioanalyzer and RNA 6000 Nano chips, with RIN values 8 or higher. RNA-seq libraries were prepared by Edinburgh Genomics from 1 µg total RNA using the Illumina TruSeq stranded mRNA-seq kit as per the manufacturer’s instructions. Libraries were pooled and sequenced to 50 base paired-end on the Illumina NovaSeq platform to a depth of ~ 46 million paired-end reads per sample. Reads were mapped to the rat reference genome using STAR RNA-seq aligner version 2.4.0i . Read counts per gene were generated from mapped reads with featureCounts version 1.6.3 , using gene annotations from Ensembl version 82 .
P60-90 male WT and Nlgn3−/y rats were anaesthetised with isoflurane and decapitated. The brain was extracted and cooled in ice-cold, carbogenated cACSF. Cortical or PAG tissue was dissected, snap frozen on dry ice, and weighed. Tissue was homogenised in ice-cold lysis buffer (150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0), protease inhibitors (Sigma), phosphatase inhibitor cocktail sets II and III (Sigma)). Samples were boiled (95 °C, 5 min) in Laemmli buffer (0.004% bromophenol blue, 10% β-mercaptoethanol, 10% glycerol, 4% SDS, 0.125 M Tris–HCl), centrifuged (16,000 G, 5 min), and vortexed.
Pierce™ BCA Protein Assay Kits (Fisher Scientific) were used to determine protein concentrations and measured using a CLARIOstar plate reader (BMG Labtech). Sample concentrations were calculated based on a bovine serum albumin standard curve (2–0.625 mg/ml).
Equal amounts of sample (20 µg total protein) along with protein ladder (PageRuler Plus Prestained Protein Ladder, Fisher Scientific, diluted in Laemmli buffer) were resolved on 10% Mini-PROTEAN TGX Precast Protein Gels (Bio-rad, 50 V 30 min, 150 V 1 h). Gels were washed in transfer buffer (Bio-rad) before transfer to nitrocellulose membranes (Bio-rad, 85 V, 2 h).
The membranes were blocked (Li-Cor buffer, 1 h) before incubation with primary antibodies (anti-NLGN3 C-terminus, Synaptic Systems, SySy-129 113, 1:1000, RRID: AB_2619816.; anti-NLGN3 N-terminus, Novus Biologicals, NBP1-90,080, 1:1000, RRID: AB_11027178) in blocking buffer with 0.01% sodium azide (10 min), then in secondary antibody (goat anti-rabbit 800, Li-Cor 1:500) in blocking buffer (2 h). After washing in TBST (TBS: Bio-rad, Tween 20: Sigma Aldrich) and TBS, membranes were imaged (Odyssey infrared, Li-COR Bioscience).
Rats aged P60-90 were used for all behaviour experiments.
Rats were placed inside a 60 × 60 cm arena with fresh bedding on the floor and white walls. The light intensity was uniformly ~ 20 lx. Animals were allowed to explore for 10 min before returning to their home cage. This was repeated for a total of 4 days.
Marble interaction task
Rats were habituated to open field (45 × 60 cm) arena with fresh bedding (2 inch) for 20 min on two consecutive days. On day 3, the rats were allowed to explore the same arena with 20 equidistantly placed opaque glass marbles (6 cm) arranged in 4 rows and 5 columns, respectively. The procedure was recorded with the overhead camera and the analysis was done using Boris v 2.98 behaviour analysis software. The light intensity throughout was uniformly maintained at 20 lx.
Auditory fear conditioning
Fear conditioning (context A, aluminium fear conditioning chamber with grid flooring, black/white horizontal-striped cue, and ~ 5 lx blue light) and recall (context B, 35 cm wide, 20 cm deep, 40 cm high arena with fresh bedding, mint odour, ~ 20 lx yellow light, and a transparent Perspex lid) took place in sound isolation cubicles (Coulbourn Instruments, Whitehall, Pennsylvania, USA).The behaviour of the animals was recorded using a video camera and a frame grabber (30 Hz sampling). The apparatus was cleaned with 70% ethanol before and after experiments.
Context habituation involved exploration of context B for 20 min on 2 consecutive days. On day 3, the rats were subjected to auditory fear conditioning in context A. After a baseline exploration time of 2 min, rats were presented with 3 pairings of conditioned stimulus (CS) (continuous tone, 30 s, 5 kHz, 75 dB) co-terminating with a scrambled foot-shock (unconditioned stimulus, US, 0.9 mA for 1 s, Habitest system, Coulbourn Instruments, Whitehall, Pennsylvania, USA). Each CS–US pairing was separated by inter-tone interval (ITI) of 1 min (modified from .
On days 4 and 5, to determine fear memory recall and extinction, rats were given 2 min to explore context B, then presented with 13 CS, with a 30 s ITI. Fear behaviour was evaluated during pre-tone, tone, and ITI.
Contextual fear conditioning
Rats were introduced to context A and given 2 min to explore. They were then presented with 3 unconditioned stimuli (US) pairings (0.9 mA scrambled foot-shock for 1 s), with a 90 s ITI. The following day, rats were reintroduced to context A for 10 min and fear behaviour was scored.
Active place avoidance
The rotating platform (Biosignal group, Brooklyn, USA) has a rectangular grid floor 100 × 100 cm) connected to a constant DC current source box for shock delivery. This was on a circular aluminium base (90 cm above ground) and run by an arena motor. A circular fence made of transparent Perspex surrounded the platform (diameter: 77 cm, height: 32 cm). For data shown in Fig. 3C–K, a transparent lid was placed on top of the circular fence. The delivery of foot-shocks (0.2 mA, 500 ms, 1500 ms interval) was tracking based (Carousel Maze Manager ). The 60° shock-zone was located on either North or South region and counterbalanced between rats. External to the arena, 3-dimensional cues were located at different distances from the apparatus.
Rats were held in a cabinet for 30 min before experimentation. They were habituated to the rotating arena  (1.5 RPM, 2 trials, 10-min interval in opaque bucket). The following day, rats were given two training sessions over two consecutive days (8 trials per session, 10-min intervals) in which the shock-zone was active. On day 4 a single probe trial was given to animals without shock-zone to assess their avoidance memory.
An overhead ceiling camera (Firewire) connected to a frame grabber (DT3155) recorded and digitised analogue video, feeding it to the tracker software (Biosignal group, Brooklyn, USA). Post-acquisition, files were analysed in Track Explorer software package (Biosignal group, Brooklyn, USA).
Rats were placed within context A from the fear conditioning task. The rats were given 2.5 min to explore their environment, then were presented with 3 scrambled foot-shocks (0.06 mA, 1 s, 1.5-min intervals). After a further 1.5-min interval, a further 3 scrambled foot-shocks were given with the intensity increased to 0.1 mA (1 s, 1.5-min intervals). This was repeated with the foot-shock intensity increasing in increments (0.2, 0.3, 0.5, 0.7, 1 mA). Following this, after another 1.5-min interval the foot-shock amplitude was then dropped back to 0.1 mA and again 3 scrambled foot-shocks were given (1 s, 1.5-min intervals). Paw withdrawal, backpedalling, forward or backward running, and jumping behaviours were quantified.
Thermal sensitivity was assessed using tail-flick analgesia meter (Columbus Instrument). The rats were habituated to the polycarbonate restrainer for 10 min/3 days. On the 4th day, the rats were placed on the analgesia meter platform and their tail was placed in the heat slot. The heat lamp intensity was set according to the titration at various heat intensities and was fixed at 6 to get a fast response without physically damaging the tissue. Five trials were given with inter-stimulus interval of 1 min. Latency to flick the tail was documented over 5 trials.
In vivo recording/stimulation of the PAG
Implantation of local-field potential electrodes or stimulating electrodes
P60-90 rats were anesthetised with a mixture of isoflurane and O2 and their head shaved and sterilised. Each animal was placed on a heat-mat (37 °C) then mounted in a stereotaxic apparatus using atraumatic ear bars. Viscotears™ was applied to the eyes and 4 mg/kg Rimadyl analgesic injected subcutaneously. Surgery was then performed under aseptic conditions. Paw withdrawal reflexes were checked regularly throughout the surgery and level of isoflurane adjusted accordingly.
A midline scalp incision was made, and craniotomies performed to allow electrode implantation in the PAG (approximate coordinates: bregma −7.46 mm, ventral 4.2 mm, 1 mm lateral from midline). Recording electrodes (made in-house, ~ 0.5 mm, 140 µm diameter Teflon coated stainless-steel, A-M systems, USA) or bipolar stimulating electrodes (MS303/3-B/SP, Bilaney Ltd.) were stereotaxically lowered through the craniotomy(ies) to the PAG.
Recording electrodes were implanted unilaterally and affixed to skull using UV-activated dental cement (SpeedCem, Henry Schein), SuperBond (SunMedical, Japan), and dental cement (Simplex Rapid, Kemdent, UK) then connected to an electronic interface board (EIB 16, Neuralynx). Four screws (Screws and More, Germany) were attached to the skull for additional support and to serve as recording ground. Stimulation electrodes were implanted bilaterally and secured to the skull using the same methods as for recording. The incision was closed using absorbable surgical sutures and sterilised with iodine. Rats were left to recover for a minimum of 1 week prior to experiment start.
LFP recordings during fear conditioning
Recordings were made via a 16-channel digitising headstage (C3334, Intan Technologies, USA) connected to a flexible tether cable (12-pin RHD SPI, Intan Technologies, USA), custom built commutator, and Open Ephys acquisition board (OEPS, Portugal). LFP signals were bandpass-filtered from 0.1 to 600 Hz and sampled at 2 kHz in Open Ephys software. Rats implanted with LFP electrodes underwent auditory fear conditioning as described above. However, a tone habituation session of three 30 s tones (5 kHz, 75 dB, 1-min intervals) was also added before conditioning, in order to observe if ERPs were present to an unconditioned tone (NB. LFPs were only recorded during tone habituation in subset of animals (WT n = 5, KO n = 7)). Video recordings were made using Freeze Frame software (15 frames per second, Actimetrics) synchronised with electrophysiological signals using TTL pulses.
In vivo PAG stimulation
Rats implanted with stimulating electrodes were placed inside context B arena as described for the fear conditioning paradigm. Rats were allowed to explore the arena for 2 min, then stimulation (0.1 ms pulses, 100 Hz, 2 s) began at an intensity of 30 μA (DS3 isolated constant current stimulators, Digitimer Ltd.) and increased in 5 μA steps up to a maximum of 75 μA , with intervals of 3 min. Behavioural responses were recorded throughout the protocol using Freeze Frame software.
A M500-384 USB Ultrasound Microphone ultrasound detector positioned above the stimulation arena coupled to BatSound Touch Lite (Pettersson Elektronik) was used to record USVs. Recordings were sampled at 384 kHz, with a spectrogram window size of 512.
Following behavioural testing, rats implanted with recording or stimulating electrodes were anesthetised with gaseous isoflurane and intraperitoneal injection of pentobarbital (27.5 mg/kg) until hindpaw reflexes were absent. A current pulse of 100 µA for 2 s (DS3 isolated constant current stimulators, Digitimer Ltd.) was passed through the headstage to lesion electrode sites. Rats were then transcardially perfused with phosphate-buffered saline, followed by 4% paraformaldehyde (PFA). The brains were extracted and left in 4% PFA for 24 h. Brains were then cut into 80 µm sections on a vibratome or freezing microtome, and these sections mounted onto glass slides. Sections were then stained with cresyl violet acetate, covered with DPX mounting medium and coverslipped. A Leica DMR upright bright-field microscope was used to image the lesion site. Location of the lesion site was projected onto a schematic of the PAG.
Ex vivo whole-cell patch-clamp recordings
Acute brain slices were made from rats aged 4–6 weeks (or 8–10 weeks, Additional file 1: Fig. S6 only), as previously described . The brain was quickly extracted and cooled in ice-cold (> 4 °C) carbogenated (95% O2/5% CO2) cACSF. The cerebellum was removed, and the brain cut coronally in half before slicing the PAG coronally at 0.05 mm/s into 400 µm slices on a Leica VT 1200S vibratome. Slices were allowed to recover in carbogenated cACSF at 35 ± 1 °C for 30 min, and then stored at room temperature until recording.
Slices were transferred to a recording chamber where they were perfused with carbogenated recording-ACSF (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 25 mM glucose, 1 mM MgCl2, 2 mM CaCl2) at 31 ± 1 °C at a rate of 3–6 ml/min. Slices were visualised using infrared differential interference contrast (IR-DIC) video microscopy, using a digital camera (DAGE-MTI) mounted on an upright microscope (U-CA, Olympus, Japan) and a 40 × water immersion objective was used for all experiments. These were paired with Scientifica slicescope, patchstar and heater units and controlled using LinLab 2 (Scientifica).
Electrodes with 3–6 MΩ tip resistance were pulled from borosilicate glass capillaries (1.7 mm outer/1 mm inner diameter, Harvard Apparatus, UK) horizontal electrode puller (P-97, Sutter Instruments, CA, USA). A potassium-gluconate based internal solution (120 mM K-gluconate, 20 mM KCl, 10 mM HEPES, 4 mM NaCl, 4 mM Mg2ATP, 0.3 mM Na2GTP, pH 7.4, 290–310 mOsm) was used for all current-clamp recordings. A caesium-gluconate based internal solution (140 mM Cs-gluconate, 3 mM CsCl, 10 mM HEPES, 0.2 mM EGTA, 5 mM QX-314 chloride, 2 mM MgATP, 0.3 mM Na2GTP, 2 mM NaATP, 10 mM phosphocreatine, pH 7.4, 290–310 mOsm) was used for all voltage-clamp recordings.
Cells in the dorsal and ventral PAG were identified by area. A −70 mV holding potential was applied following the creation of a > 1 GΩ seal. The fast and slow membrane capacitances were neutralised before breaking through the cell membrane to achieve whole-cell configuration. For mEPSC recordings, gap-free recordings were performed in voltage-clamp configuration for 10 min in the presence of picrotoxin (50 µM) and tetrodotoxin (300 nM). Cells were discarded if access resistance was > 30 MΩ or changed by > 20%. Intrinsic property recordings were carried out in current-clamp configuration, as follows. Resting membrane potential (RMP) of the cell was recorded with current clamped at 0 pA, and all other protocols recorded with appropriate current injection to hold the cell at −70 mV. Cells were discarded if RMP was more depolarised than −40 mV or if access resistance was > 30 MΩ or changed by > 20%. Input resistance and membrane time constant were assessed by injecting a −10 pA step, and cell capacitance calculated from these values. Input–output curves and rheobase potential were assessed by current injections of −200 to + 100 pA for 500 ms (10 pA steps). Action potential kinetics were gleaned from the rheobase action potential. Recordings were made using a Multiclamp 700B amplifier linked to pCLAMP™ Clampex software (Molecular Devices). Signals were sampled at 20 kHz (Digidata1440 or Digidata1550A, Molecular Devices) and Bessel-filtered at 2 kHz for voltage-clamp recordings and 10 kHz for current-clamp recordings.
Quantification and statistical analysis
Fear behaviour was scored as either “classic freezing”, defined as no movement except for respiration , or “paw immobility response”, defined as all 4 paws unmoving, however allowing for movement of the head and neck. Behaviours were scored if lasting > 1 s. For the shock-ramp paradigm, paw withdrawal responses, backpedalling, forward/backward running, and jumping were scored. For dPAG stimulation experiments, response behaviours were scored as freezing, startle, attention, running, or jumping, according to criteria described previously . All behaviour was manually scored using BORIS  or in-house software Z-score (created by O. Hardt).
Stimfit software  combined with custom-written Matlab scripts (A. Jackson) were used for whole-cell patch-clamp data analysis. mEPSCs were analysed for the final 3 min of the 10-min recording. Events were detected using template-matching and filtered to 3 × standard deviation of baseline .
Data collected from LFP recordings were analysed using custom-written MATLAB scripts (F. Inkpen, A. Jackson). Raw traces of 3 tones were averaged, and then z-scored to normalise data to baseline noise. Peak and trough of the LFPs were manually selected.
Raven Lite software (Cornell Lab, Centre for Conservation Bioacoustics) was used to generate spectrograms and to manually quantify USVs.
Throughout, all data is shown as mean ± SEM, or as percentages where appropriate. Statistics were carried out using GraphPad Prism software 8.0, SPSS, or RStudio. Two-way ANOVAs with Holm–Sidak post hoc repeated measures test (Figs. 2, 3, 4, 5, 6, 7, Additional file 1: Figs. S1, 2, 3, 5–7), Pearson’s R correlation (Additional file 1: Fig. S7), unpaired t-tests (Fig. 4, Additional file 1: Fig. S4), paired t-tests (Additional file 1: Fig. S4), Fisher’s exact tests (Figs. 3, 7), three-way ANOVAs (Figs. 2, 6, Additional file 1: Fig. S1), or generalised linear mixed modelling (GLMM) (Fig. 5, Additional file 1: Fig. S5) were employed. N was taken to be animal average in all cases to avoid pseudoreplication, except for when GLMM statistical analysis was employed. R packages lme4 and car were utilised to perform GLMMs. p values of < 0.05 were taken to be significant, and one star (*) represents all p values < 0.05 throughout. Full details of statistical tests and results are described in Additional file 2: Tables S1, 2.