B6;129-Nlgn3tm1Sud/J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and crossed onto a C57Bl/6 background for over 10 generations (F10). NL3^R451C and wild-type (WT) animals were derived by mating heterozygous females with NL3^R451C males, which produced 50:50 WT and NL3^R451C offspring. Only male (Y/+ and Y/R451C) mice were used for this study. All electrophysiology experimental protocols were approved by the Florey Institute of Neuroscience and Mental Health Animal Ethics Committee and the animal behavior and autoradiography experimental protocols were approved by the Monash Institute of Pharmaceutical Sciences (MIPS) Animal Ethics Committee.

Following anesthesia with 1%–3% isoflurane (inhalation), postnatal day 15–46 mice were decapitated and cortical slices were cut (300 μm thick). Using whole cell patch clamp technique in voltage clamp mode either miniature inhibitory or excitatory postsynaptic currents (mIPSCs/mEPSCs) were recorded from pyramidal-like neurons (selected based on their large soma size and morphology as visualized via infrared differential interference contrast optics) of the basolateral amygdala.

Brain slices were bathed in artificial cerebrospinal fluid containing 125 mM NaCl, 25 mM NaHCO₃, 3 mM KCl, 1.25 mM NaH₂PO₄H₂O, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, aerated with 95% O₂/5% CO₂ to a final pH of 7.4. TTX (0.5 mM) was present in all recordings and 50 μM D-AP5 (Tocris), 10 μM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris) and 50 μM Picrotoxin (Tocris) were added for inhibitory and excitatory recordings respectively. Internal pipette solutions were 120 mM CsCl, 10 mM Hepes, 5 mM NaCl, 1 mM MgCl₂, 0.3 mM NaGTP, 3 mM MgATP, 10 mM EGTA, 5 mM QX-314, 8 mM biocytin hydrochloride for inhibitory, and 117.5 mM CsMeSO₄, 10 mM Hepes, 10 mM tetra-ethylammonium chloride (TEA-Cl), 15.5 mM CsCl, 1 mM MgCl₂, 10 mM Na phosphocreatine, 8 mM NaCl, 0.3 mM NaGTP, 4 mM MgATP, 5 mM EGTA, 1 mM QX-314, 8 mM biocytin hydrochloride for excitatory recordings.

For each cell, synaptic events were detected using the template matching algorithm of pClamp 9.0 (Molecular Devices) for 3 × 3 min recordings held at a membrane potential of −70 mV and at a constant temperature of 32°C. Data were only included in analysis if the series resistance was <25 MΩ and did not change by >20% during the course of the recordings. Using analysis software (Clampfit, Molecular Devices, San Jose, CA, USA) the peak amplitude and inter-event interval of these post synaptic currents were measured and compared. The results were plotted as a cumulative fraction curve allowing the average population of events over a 3 min recording to be shown. Data was also compared using mean values over the entire 3 min recording period and plotted as a box plot.

In order to confirm the approximate location of each cell recorded an image of the patch pipette and whole-cell was acquired using a Sony (CCD XC-ST50CE) camera attached to an Olympus BX-51 microscope. These images were overlaid onto a plate image from the Mouse Brain Atlas (Paxinos and Franklin, 2001) corresponding to figure 42 (Bregma −1.34 mm, interaural 2.046 mm) using the imaging software Corel Draw Graphic Suite X7.

Mice were weaned from their mother at postnatal day 21 and allowed to habituate to a 12 h dark/light cycle (lights on at 2200, lights off at 1000) in the holding room for at least 7 days before initiation of juvenile social interaction testing at postnatal day 28 ± 3. Locomotor activity testing was conducted at postnatal day 56 ± 5, and resident intruder testing was conducted at postnatal day 73 ± 7.

At postnatal day 28, each mouse was isolated in a clean cage in a separate room 1 h prior to testing. Mice were paired with littermates or non-littermates based on weight and placed in opposite corners of a 39.5 × 39.5 cm perspex arena lined with fresh sawdust bedding. Interactions between WT-WT, NL3-WT and NL3-NL3 pairs were assessed by counting the number of incidences of the following behaviors: anogenital sniffing, head sniffing, self-grooming (grooming for a minimum of 2 s), jumping and rearing and were defined as per Chadman et al. (2008).

Each mouse was tested for juvenile social interaction once, with the exception of one mouse that was paired with two different mice for separate interaction recordings on different days. Behavior was recorded for 10 min, as reported previously (Chadman et al., 2008). Between each test, sawdust bedding in the arena was disposed of and replaced with fresh bedding. Recordings of mouse behaviors were assessed manually by a single observer blinded to genotype and treatment.

Using the well-established Resident-Intruder assay (Miczek et al., 2001; Koolhaas et al., 2013), territorial aggression behaviors were assessed by monitoring aggressive behavior displayed by the resident (subject mouse) when a novel juvenile mouse was placed into the resident’s home cage for a period of 5 min.

At postnatal day 66 ± 7 resident mice (i.e., WT or NL3) were habituated in single cages. C57Bl/6 WT intruder mice were obtained from Monash Animal Services (Monash University, Clayton) on the same day as resident mice to allow 7 days of habituation into the dark/light cycle of the holding room before testing. To differentiate intruder mice from residents, a small patch of fur on their upper back was shaved using animal clippers.

At postnatal day 73 ± 7, following 7 days of social isolation, resident mice were weight-matched with a younger intruder mouse at an age of postnatal day 45–56. In order to minimize influences of variable weight between juvenile intruders and resident mice, weights were taken into consideration when allocating pairs. Weight matching was achieved with a mean weight difference of 2 g. Resident mice were paired with the same intruder mice for four consecutive days. Resident mice were injected with either vehicle (20% captisol^®, a polyanionic cyclodextrin derivative in water) or the CB1 receptor agonist WIN55,212-2 at either 0.3 or 1 mg/kg 20 min before testing. The intruder mouse was then placed into the home cage of the resident and interactions were recorded for 5 min by video camera. Neither the genotype nor the drugs administered were known to the observer at the time of video analysis. Parameters scored were: attack latency (latency to first attack), attack incidence, attack duration, and the number of occurrences of anogenital sniffing, head sniffing, mounting and number of tail rattles. An attack was recorded when one mouse approached the other mouse at high speed to bite (usually on the back) and grasped the mouse using forepaws, sometimes rolling over multiple times in order to maintain contact while biting.

Cumulative curves describing the distribution of miniature synaptic events (amplitude and inter-event interval) were compared using the non-parametric Kolmogorov-Smirnov statistical test (GraphPad Prism software). Mean values for amplitude and inter-event interval for miniature synaptic events for each genotype were also compared using a Student’s t-test. One-way ANOVA was used to analyze juvenile social interaction data and the Holm-Sidak’s multiple comparison test was used to differentiate significance between groups. Locomotor activity assessment (LMA) and aggression test parameters were analyzed using two-way ANOVA. When a main effect was detected, a Tukey’s multiple comparisons test was conducted to evaluate significant differences.

Synaptic currents were recorded from pyramidal-like neurons of the amygdala in the NL3^R451C and WT littermate controls. The age of the animal was not found to impact the post synaptic recordings (data not shown) so data was pooled. Raw traces of mEPSCs from WT and NL3^R451C mice demonstrated an increase in event amplitude in mutant mice (Figures 1A,B). A comparison of cumulative fraction amplitude curves showed larger amplitudes for NL3^R451C mice than WT littermates (Figure 1C; P = 0.0096). No differences in the inter-event interval in NL3^R451C and WT mice were observed (Figures 1D,F). In agreement with the analysis of cumulative fraction curves, the mean amplitude of events in NL3^R451C mice was larger (Figure 1E; P = 0.016). The location of each recorded cell is shown overlaid on an image of a coronal section from the Mouse Brain Atlas (Paxinos and Franklin, 2001; Figure 1G).

Inhibitory currents had reduced amplitude and an increased inter-event interval in NL3^R451C mice (Figure 2) as shown by representative raw traces (Figures 2A,B) and cumulative fraction curves (Figures 2C,D). The mIPSC peak amplitude in neurons from NL3^R451C mice was significantly smaller than in WTs (Figure 2C; P = 0.0008). When analyzed using the Kolmogorov Smirnov test, inter-event interval was increased in NL3^R451C mice (Figure 2D; P = 0.001). The mean event amplitude was significantly smaller in NL3^R451C compared to WT littermates (Figure 2E; P = 0.005). When comparing mean values, the inter-event interval was unchanged between genotypes (Figure 2F). The location of each recorded cell is shown in Figure 2G (inset).

To determine if behavioral phenotypes including aggression caused by the R451C mutation penetrate across background strains we examined a range of behavioral outputs in WT and NL3^R451C mice bred on a pure C57Bl/6 background strain. Freely interacting juvenile mice (4 weeks of age) were observed for frequency of self-grooming, head-sniffing and ano-genital sniffing, jumping and rearing.

There were no significant differences observed between the WT/WT pairs and NL3^R451C/NL3^R451C pairs for self-grooming, head-sniffing, ano-genital sniffing and rearing (Figures 3A–C,E). There was a main effect of genotype on jumping behavior (F_(2,14) = 6.669; P = 0.0092; Figure 3D). Mixed genotype pairs (WT/NL3^R451C) showed an increase in jumping events compared to the WT/WT (p = 0.009) and NL3/NL3 (P = 0.0356) pairs as revealed by the Holm-Sidak’s multiple comparison test.

To investigate the possibility of NL3^R451C behavior being regulated through a CB1 receptor pathway, we tested for aggressive behaviors in mice following administration of the CB1 receptor agonist WIN 55, 212–2 (0.3 and 1 mg/kg). Aggressive behavioral parameters including the number of mounting episodes and tail rattles displayed as well as attack incidence, attack duration and attack latency were compared between NL3^R451C mice and WT littermates using the resident-intruder test (Figure 4). Overall, we identified a significant effect of genotype on all parameters reported and a drug effect for attack incidence, duration and latency (ANOVA). NL3^R451C mice exhibited more mounting behavior and tail rattles (Figure 4A) compared to WT littermate controls (P = 0.0439 and P = 0.0172 respectively). The incidence (Figure 4A) and duration of attacks (Figure 4B) were also greater in the NL3^R451C mice (P = 0.0002 and P = 0.025 respectively). Furthermore, attack latency was reduced in mutant mice (P = 0.0001) compared to WT littermates (Figure 4B). These findings suggest increased aggressive behaviors in NL3^R451C mice compared to wild-type littermates.

Administration of WIN55,212-2 (0.3 and 1 mg/kg) caused no change in the occurrence of mounting behavior or tail rattles in either the NL3^R451C or WT littermates (F_(2,19) = 1.953; P = 0.1693 and F_(2,19) = 1.103; P = 0.3521 respectively; ANOVA repeated measures; Figure 4D and data not shown). WIN55,212-2 altered attack incidence (F_(2,19) = 8.214; P = 0.0027; duration F_(2,19) = 7.73; P = 0.0035) and latency of attack (F_(2,19) = 9.79; P = 0.0012; ANOVA repeated measures). Post hoc analyses (Tukey) identified that in NL3^R451C mice, administration of WIN 55, 212–2 reduced attack incidences (P = 0.0096 for 0.3 and P = 0.0051 for 1 mg/kg), attack duration (P = 0.0125 for 0.3 and P = 0.0063 for 1 mg/kg), and increased attack latency (0.3 mg/kg P = 0.0063 and 1 mg/kg P = 0.0026; Figures 4C,E,F).

In order to clarify the potential sedative effects of WIN55,212-2 at 0.3 and 1 mg/kg, the locomotor activity was recorded and observed. There was no effect of genotype on either the track length (F_(1,36) = 0.062; P = 0.805) or average velocity (F_(1,36) = 0.0041; P = 0.949) measured. Similarly, there was no effect of drug on track length (F_(1,36) = 2.653; P = 0.084) or average velocity (F_(1,36) = 2.067; P = 0.14 respectively; Supplementary Figures S1A,B).

To further understand the behavioral differences, CB1 and GABA_A receptor radioligand binding was examined using autoradiography. Radioligand binding at these receptors was found to be normal in the prefrontal cortex (P = 0.7634), hippocampus (P = 0.6020), VMH (P = 0.9147), amygdala (P = 0.3860) and PAG (P = 0.3860; Supplementary Figures S2A,B) compared to the WT littermates. There were also no significant differences found in the density of GABA_A receptors in the prefrontal cortex (P = 0.4114), hippocampus (P = 0.0766), VMH (P = 0.1725), amygdala (P = 0.3362) and PAG (P = 0.6425; Supplementary Figures S2C,D). This suggests that the NL3 gene mutation had no effect on expression of these receptors and that the behavioral effects of the CB1 receptor agonist (WIN55,212-2; Figure 4) were not secondary to altered receptor levels.