Oxytocin normalizes altered circuit connectivity for social rescue of the Cntnap2 knockout mouse.

Experimental enhancement of NAc

OXT signalling sufficiently increases social behavior in KO mice

To directly examine the functional contribution of OXT signalling in the NAc to the pro-social effect of OXT in KO mice in vivo , we performed a home cage reciprocal social interaction assay in mice after a bilateral microinjection of TGOT (a selective OXTR agonist; Eliava et al., 2016 ; Lowbridge et al., 1977 ) into the NAc through an implanted cannula ( Figure 6A , B ; S6A ). Although both NAc core and shell areas have been implicated in in social reward learning ( Dolen et al., 2013 ; Hung et al., 2017 ), in this experiment we specifically targeted the NAc shell (NAcSh) area due to its heavy innervation with OXT fibers ( Liao et al., 2020 ), the previously demonstrated excitatory response to OXT application in vivo ( Moaddab et al., 2015 ), and the proposed role for the NAcSh as a social hub region ( Johnson et al., 2017 ). In a separate trial, each mouse received an equal amount of saline infusion as control experiments. We found that KO mice spent a significantly longer amount of time investigating a novel, sex-matched juvenile mouse after receiving TGOT, when compared to the saline trials (n=10 mice, p=0.04; Figure 6B ). These results demonstrate that OXTR activation in the NAcSh is sufficient to increase social interaction in KO mice. Finally, we asked whether the pro-social effects of OXT in KO mice could simply be replicated by triggering local endogenous OXT release within the NAcSh. To test this, we used an optogenetic strategy wherein expression of ChETA, a variant of Channelrhodopsin with fast time kinetics ( Gunaydin et al., 2010 ), was targeted to PVN OXT neurons ( Figure 6C ). Histological examination of these mice confirmed robust expression of eYFP in the PVN and OXT fibers in the NAcSh ( Figure 6D ), as well as proper targeting of the implanted cannulae ( Figure S6B ). ChETA and control mice were tested with the home cage reciprocal social interaction assay, with or without blue light (457 nm) stimulation throughout the entire experimental period (10 minutes). We observed that light stimulation significantly increased the social behaviour of KO-ChETA mice when compared to trials performed without light stimulation (p=0.03; Figure 6E ). We did not observe a similar effect in control KO mice (p=0.75; Figure 6E ), confirming that the behavioural change induced by blue light was driven by optogenetic excitation of OXT neuron terminals. These two lines of independent evidence, pharmacological and optogenetic, establish that OXT signalling in the NAcSh serves as an important modulator likely mediating the pro-social effect of exogenous and endogenous OXT in KO mice.

STAR Methods Resource Availability: Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Daniel H. Geschwind ( dhg@mednet.ucla.edu ) Materials Availability This paper does not report original materials.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental Model

Mutant mice lacking the Cntnap2 gene (Caspr2 - null mice or Cntnap2 −/− ) were obtained from Dr. Elior Peles ( Poliak et al., 2003 ) and backcrossed to the C57BL/6J background for over 12 generations. Experimental Cntnap2 mutant and WT mice were obtained from heterozygous crossings and used for experiments when they reached adulthood (8 weeks or older). Both sexes were used as experimental animals – they were randomly assigned to groups and no obvious differences were noted. The three obtained genotypes were housed together with three to four same sex mice per cage. A subset of experimental mice were obtained from crossing male breeder mice that are heterozygous for Cntnap2 KO and homozygous Oxt-Cre (obtained from JAX, #024234) with female heterozygous Cntnap2 KO mice to produce offspring experimental mice that are homozygous for Cntnap2 KO and heterozygous for Oxt-Cre (Cntnap2 −/− ;Oxt Cre/+ ). All mice were kept in 12 hr light/12 hr dark cycle and had ad-lib access to food and water. All procedures involving animals were performed in accordance with the UCLA Animal Research Committee, and the NIH Guide for the Care and Use of Laboratory Animals.

Method Details Magnetic resonance imaging

The mouse to be scanned was first briefly anesthetized with 2% isoflurane vaporized in oxygen flowing at 1 L/min, then placed on an MRI-compatible cradle. A single-channel surface coil (Bruker) was placed over the head of the mouse. For resting-state scans, a 3 mm-thick agar gel cap (Sigma, 3% in distilled water) was placed between the head and the surface coil, in order to reduce signal distortion in the blood-oxygen-level-dependent (BOLD) signal ( Adamczak et al., 2010 ). To minimize time-dependent effects of the anesthetic on the BOLD signal, these initial steps were performed within a 10–15-minute time window ( Magnuson et al., 2014 ). Isoflurane was gradually discontinued and sedation was initiated with a single subcutaneous (s.c.) injection of dexmedetomidine (Dexdomitor®, Zoetis; 0.15 mg/kg) followed by continuous s.c. infusion at 0.3 mg/kg/hr throughout the duration of the imaging as described in a previous publication ( Adamczak et al., 2010 ). Respiration and body temperature of the mouse were continuously monitored remotely, and maintained in a physiological range (37 ± 1 °C; Small Animal Instruments Inc.) by a homeothermically-controlled forced warm air over the body (SA11 Instr, Inc., USA). At the end of the imaging session, sedation was reversed by atipamezole (Antisedan®, Pfizer) at 1.5 mg/kg (i.p.). Magnetic resonance imaging was performed using a 7 Tesla (T) Biospec small animal MRI system using Paravision 5.2 software (Bruker). Data were acquired using the S116 Bruker gradients (400 mT/m) in combination with a single-channel surface coil (described above) and a 72 mm birdcage transmit coil. An initial series of scans was performed to confirm proper head position, then localized FASTMAP shimming was performed to improve field homogeneity. pharmacological MRI T2-weighted structural scans were acquired with a Rapid-Relaxation-with-Enhancement (RARE) sequence (RARE factor=8, Echo time (TE)=56 ms, repetition time (TR)=6,018 ms, 4 averages, field-of-view (FOV)=1.8× 1.2cm, slice thickness=0.5 mm, 18 slices, FA=90 deg, bandwidth (BW)=50 kHz, matrix=60×40). Then, 45-minute long functional (BOLD) data were acquired using the same image geometry as the structural scans, with a one-shot, interleaved, gradient-echo echo planar imaging sequence with the following parameters: TE=16 ms, TR=2,000 ms, FA=70 degrees, BW 400 kHz and a data matrix of 60×40). 10 dummy scans were used to allow the T1 signal to reach steady-state prior to signal acquisition. OXT (Sigma, 1 mg/kg i.p at 1 mg/mL) or saline was administered following 5 minutes of baseline imaging data collection, after which imaging data was acquired in a continuous manner for the next 40 minutes. Resting-state MRI Structural and functional data were acquired using above-described parameters, with the following modifications: field-of-view (FOV)=3.0×3.0 cm, matrix=128×64; functional (BOLD) scans acquired using a two-shot gradient-echo echo-planar imaging (GE-EPI) sequence, TE=19 ms, TR=1,000 ms (2,000 ms per volume), FA=70 degrees, BW 400 kHz and a data matrix of 128×64, Fourier transformed to 128×128, voxel resolution 0.23×0.23×0.5 mm). For each mouse, one 5-minute resting-state scan (150 volumes) was acquired at the baseline, and then OXT (Sigma, 1 mg/kg i.p. at 1 mg/mL) or saline of the same volume was administered via a catheter placed prior to scanning. 5-minute resting-state scans were acquired every 10 minutes, during the 40-minute period following OXT/saline administration. OXT receptor autoradiography Following anesthesia and decapitation, brains were quickly removed and frozen in −70°C isopentane, and stored at −80°C. Sets of coronal brain sections (20 μm) were cut in a cryostat and thawmounted on Superfrost plus slides (stored at −80°C until processing for autoradiography). Autoradiography was performed using 40 pM concentration of OXT receptor ligand, 125I-ornithine vasotocin (OVT; PerkinElmer, NEX254) following standard protocols ( Ferretti et al., 2019 ; Menon et al., 2018 ). Briefly, sections were thawed at room temperature, and fixed in 0.1% paraformaldehyde. After wash in 50 mM Tris-HCl (pH 7.4), the sections were incubated in 50pM 125I-labeled ligand in 50 mM Tris, 10 mM MgCl2, 0.1% bovine serum albumin (BSA), and 0.05% bacitracin. Excess ligand was removed by washing in 50 mM Tris-HCl (pH 7.4) / 10 mM, then in 50 mM Tris-HCl (pH 7.4) / 10 mM MgCl2. Finally, the sections were air-dried and exposed to Kodak BioMax MR film. Receptor binding patterns were qualitatively assessed. iDISCO+ brain-wide imaging Sample processing and imaging Animals were single housed for 2 hours prior to OXT administration (Sigma, 1 mg/kg i.p). They were returned to their single-housed cages immediately after injection. 2 hours after injection, they were perfused transcardially with PBS followed by 4% PFA in PBS. Brains were extracted and postfixed overnight at 4°C. The brains were then divided into two halves by cutting in the sagittal plane, then processed with the iDISCO+ immunolabeling, following a previously described protocol ( Renier et al., 2016 ). Samples were stained with c-Fos primary antibody (Synaptic Systems 226 003, 1:500), then Alexafluor 647 secondary antibody (ThermoFisher Scientific, 1:500). At least one day after clearing, iDISCO+ samples were imaged on a light-sheet microscope (Ultramicroscope II, LaVision Biotec) equipped with a sCMOS camera (Andor Neo) and a 2Å~ /0.5 NA objective lens (MVPLAPO 2Å~) using 488-nm and 640-nm lasers. The samples were scanned with a z-thickness of 3 μm using the continuous light-sheet scanning method with the dynamic horizontal focus for the 640-nm channel (20 acquisitions per plane), and without horizontal scanning for the 488-nm autofluorescence. Modifications and continuous updates to the iDISCO+ protocol can be found at http://www.idisco.info . Chemogenetic activation of endogenous OXT release Bilateral stereotaxic injections of 500 nL of AAV2-mOXT-hM3D(Gq)-mCherry-WPRE (custom generated from Signagen with plasmid from D. Geschwind laboratory ( Penagarikano et al., 2015 ), or AAV2-hSyn-DIO-hM3D(Gq)-mCherry ( Krashes et al., 2011 ) were performed into the PVN of the hypothalamus in WT and KO mice according to the following coordinates from Bregma: antero-posterior −0.60 mm; dorso-ventral −4.80 mm; lateral+/−0.20 mm. Another group of WT and KO mice received injections of AAV2-mOXT-Venus (V. Grinevich laboratory, Germany; Grinevich, 2016 ; Knobloch et al., 2012 ) or AAV2-hSyn-DIO-mCherry ( Krashes et al., 2011 ) to control for the effects of surgeries and CNO administration. Mice who received injections of the viruses containing the Double-Floxed Inverted Open reading frame (DIO) were also heterozygous for the Oxt-Cre transgene (Oxt Cre/+ ). Injections were performed at a rate of 100 nL/min with a glass micropipette (50 μm tip diameter) using a Nanoliter 2000 microinjection system (WPI) attached to a Micro4 pump (WPI). Three weeks after stereotaxic viral injections, WT and KO mice were singly housed and given clozapine-n-oxide (Enzo Life Sciences, 5 mg/kg, i.p.) after a habituation period of at least 1 hour. 2 hours after injection, mice were deeply anesthetized with 4% isoflurane and intracardially perfused with 4 % paraformaldehyde 0.1M phosphate-buffered saline (freshly diluted from 16 or 32% stock, Electron Microscopy Sciences). Brains were subsequently removed and incubated in 0.1 M phosphate-buffered solution containing 30 % sucrose at 4 °C for 2–3 days. Brains were then embedded in optimal cutting temperature solution (TissueTech) at −80°C, and cryosectioned at 50 μm thickness. Sections were stained with rabbit polyclonal anti-c-Fos antibody (1:500, Santa Cruz, sc-52) and goat anti-rabbit Alexa 647 secondary antibody (1:500). Confocal images were obtained at 10x magnification using a Zeiss 880 laser-scanning confocal microscope and analyzed using ImageJ (NIH).

Home cage social interaction test

Home cage social interaction assay of adult KO mice was performed adapting a previously used juvenile social play protocol (Peñagarikano et al., 2015). Briefly, adult KO mice and another group of juvenile (3–5 weeks old) WT mice, serving as novel social stimuli, were each singly housed in a new complete cage set up. They were then allowed to habituate for at least 1 hour in a dark room with white noise. Immediately prior to the social interaction assay, each pair of sex-matched, juvenile-adult pair separately underwent another phase of 10-minute solitary home cage exploration period in new home cages without lids. The pair was then placed together into another new home cage with no lid, meeting each other for the first time. They were allowed to freely interact for 10 minutes. In vivo infusion of TGOT into the NAc shell Adult KO mice were implanted with bilateral guide cannulae (Plastics One; 26G, 1.0 mm spacing, 3.5 mm long) targeting the NAcSh using the following stereotaxic coordinates from Bregma: anterio-posterior +1.3 mm; dorso-ventral −3.5 mm; lateral +/−0.5 mm. After at least 4 days of recovery, experimental mice were subjected to home cage social interaction tests described as above. Prior to the 10-minute solitary home cage exploration period, TGOT (0.25 μL/side for a total of 0.5 μL/mouse, dissolved at 14 ng/μL in 0.9% saline; Phoenix Pharmaceuticals) or an equivalent volume of saline was delivered to each side of the NAcSh for a total of 0.5 μL injection volume per mouse. Infusion was delivered a rate of 0.1 μL/min via a bilateral internal cannula (Plastics One; 33G, 1.0 mm spacing, 4.5 mm long). The internal cannula was left in place for 2.5 minutes after injection to allow for diffusion into the surrounding brain tissue. Each mouse underwent both TGOT and saline infusion trials separated by at least 1 day, the order of which were counterbalanced within the experimental group. After the conclusion of behavior experiments, mice were injected with a small volume (~0.1 μL) of 1% Evans blue (Sigma; diluted to 1% in 0.9% saline) through the internal cannula for visualization of injection sites, then perfused with paraformaldehyde solution (4% in PBS, Wako Chemicals). The brains were extracted, vibratome-sectioned and imaged on a confocal microscope (LSM 900, Zeiss) to confirm cannula placement.

Optogenetic stimulation of OXT release in the NAcSh Adult

KO mice were stereotaxically injected AAV1-Ef1a-DIO ChETA-EYFP (500 nL/side; Addgene #26968; Gunaydin et al., 2010 ) into the PVN of the hypothalamus as described above. Another group of KO mice were injected with or AAV2-CAG-Flex-eGFP-WPRE (Addgene #51502; Oh et al., 2014 ) or AAV2-mOXT-Venus (generated by V. Grinevich laboratory, Germany; Grinevich, 2016 ; Knobloch et al., 2012 ) to serve as controls. Following viral injections, dual-core optic cannula (200 μm core, 700 μm spacing, 0.39 NA, Thorlabs) was implanted into the NAcSh of each mouse using the following stereotaxic coordinates from Bregma: antero-posterior +1.3 mm; dorso-ventral −3.5 mm; lateral +/−0.35 mm. The cannulae were fixed to the skull of mice using Metabond (Parkell). After at least 3 weeks have passed, home cage social interaction assay (described above) was performed with or without optogenetic stimulation in separate trails. A patch cord was connected to the implanted mice immediately prior to the 10-minute habituation stage in the cage without the lid. During optogenetic stimulation trials, mice received light stimulation (473 nm DPSS laser at 30 Hz, 10 ms width) at ~30 mW laser intensity measured from the tip of each dual-core cannula (~15 mW estimated laser intensity from each core) throughout the entire free social interaction period. Trials were separated by at least 1 day, and the order of the two trials were counterbalanced. After the conclusion of behavior experiments, mice were perfused with paraformaldehyde solution (4% in PBS, Wako Chemicals) The brains were extracted, vibratome-sectioned and imaged on a confocal microscope (LSM 900, Zeiss) to confirm cannula placement and PVN-specific expression of eYFP, eGFP, or Venus. A subset of sections were stained with PS38 mouse monoclonal anti-oxytocin antibody (1:500, a gift of H. Gainer; Ben-Barak et al., 1985 ) and goat anti-mouse Alexa 555 secondary antibody (1:500; Thermofisher) to confirm cellular identity.

Quantification and Statistical Analysis

Each n represents the number of animals. N values for each experimental dataset are listed in the figure legends. Summary data are either represented as box-whisker plots (whisker: min-max values, box: 25–75 th percentile) or bar plots (mean ± SEM) with individual values. Kolmogorov-Smirnov test was performed to check for normality of datasets prior to conducting t-test or ANOVA. The details of each analysis method and statistical test used are listed below.

MRI analysis

Analysis was performed using FSL tools ( Smith et al., 2004 ; see Figure S1B for an illustration of the analysis pipeline). The following pre-analysis corrections were performed: BET brain extraction, MCFLIRT motion correction, slice timing correction, and smoothing at 5 mm. Scans with a large magnitude of head movement were identified and excluded from further analysis. Using FLIRT, all structural scans were co-registered to a single structural scan selected as a reference. The same transformation matrix was then applied to functional (BOLD) scans. For Atlas-based analyses, regions-of-Interest (ROIs) were defined using a parcellated in vivo mouse brain atlas custom-constructed from the Allen brain histological atlas ( Lein et al., 2007 ) co-registered to Dorr 3D mouse brain atlas ( Dorr et al., 2008 ). The parcellated atlas was co-registered to each functional scan. 46 brain ROIs were defined intra- and inter-hemispherically: OLF, olfactory cortex, ORB, orbital area, ACC, anterior cingulate cortex, ASA, association areas, AUD, auditory cortex, INS, insular cortex, LIM, limbic cortex, MO, motor cortex, SS, somatosensory cortex, PIR, piriform cortex, RSA, retrosplenial area, DP, dorsal peduncular cortex, NAC, nucleus accumbens, LS, lateral septum, HPC, hippocampal area, BNST, bed nucleus of stria terminalis, CP, caudoputamen, LH, lateral hypothalamus, TH, thalamus, PAL, pallidum, MH, medial hypothalamus, MS, medial septum, MID, midbrain, PVN, paraventricular nucleus of the hypothalamus, RN, reticular nucleus, VTA, ventral tegmental area (L, left, R, right). Grouping of major brain structures (CTX, HPC, MID, THAL, HYPO, FN, OLF) followed the annotation rules of the Allen Brain Atlas ( Lein et al., 2007 ; Wang et al., 2020 ). Classification of “social” (OLF, ORB, ACC, INS, LIM, PIR, DP, NAC, LS, BNST, LH, MH, MS, and PVN) and “other” regions (the rest of ROIs) was performed based on established roles evident in existing literature (e.g. Anderson, 2016 ; Chen and Hong, 2018 ; Kim et al., 2015 ). Pharmacological MRI Statistically significant BOLD activation associated with OXT or saline administration was first identified at the single-subject level using general linear modeling (GLM; cluster thresholds: z