Acute, chronic and conditioned effects of intranasal oxytocin in the mu-opioid receptor knockout mouse model of autism: Social context matters

Abstract

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behaviours. Multiple studies have highlighted the potential of oxytocin (OT) to ameliorate behavioural abnormalities in animal models and subjects with ASD. Clinical trials, however, yielded disappointing results. Our study aimed at assessing the behavioural effects of different regimens of OT administration in the Oprm1 null mouse model of ASD. We assessed the effects of intranasal OT injected once at different doses (0.15, 0.3, and 0.6 IU) and time points (5, 15, and 30 min) following administration, or chronically, on ASD-related behaviours (social interaction and preference, stereotypies, anxiety, nociception) in Oprm1^+/+ and Oprm1^-/- mice. We then tested whether pairing intranasal OT injection with social experience would influence its outcome on ASD-like symptoms, and measured gene expression in the reward/social circuit. Acute intranasal OT at 0.3 IU improved social behaviour in Oprm1^-/- mice 5 min after administration, with limited effects on non-social behaviours. Chronic (8-17 days) OT maintained rescuing effects in Oprm1 null mice but was deleterious in wild-type mice. Finally, improvements in the social behaviour of Oprm1^-/- mice were greater and longer lasting when OT was administered in a social context. Under these conditions, the expression of OT and vasopressin receptor genes, as well as marker genes of striatal projection neurons, was suppressed. We detected no sex difference in OT effects. Our results highlight the importance of considering dosage and social context when evaluating the effects of OT treatment in ASD.

Fig. 1. Acute per nasal administration of OT dose-dependently restored social behaviour in Oprm1 null mice.

a Oprm1^+/+ and Oprm1^-/- mice received OT or vehicle (4 males – 4 females per genotype and treatment), via per nasal route, 5 min before the direct social interaction test and at the dose of 0, 0.15, 0.3, or 0.6 IU. In this test, vehicle-treated Oprm1^-/- mice displayed a deficit in social interaction; this deficit was partially reversed for the OT dose of 0.15 IU, fully relieved at 0.3 IU, and remained unchanged at 0.6 IU (mean duration of nose contacts: H_7,64 = 53.13, p < 0.0001, mean duration of paw contacts: H_7,64 = 49.93, p < 0.0001, number of following episodes: H_7,64 = 37.57, p < 0.0001, grooming after social contact: H_7,64 = 49.76, p < 0.0001). b When administered 15 min before testing (4 males – 4 females per genotype and treatment), the optimal dose of 0.3 IU had only partial effects on the duration of nose contacts (H_3,32 = 26.28, p < 0.0001) in Oprm1^-/- mice and no effect on the duration of paw contacts (H_3,32 = 26.59, p < 0.0001). c When administered 30 min before testing (4 males – 4 females per genotype and treatment), per nasal OT at 0.3 IU was ineffective in relieving social interaction deficit in Oprm1 null mice. d The non-peptide OT antagonist LIT183 (see Supplement 1) or its vehicle (doses of 0, 7.5, or 15 mg/kg) were administered intraperitoneally 25 min before per nasal OT administration (0.3 IU) and 30 min before direct social interaction test (4 males – 4 females per genotype, LIT183 doses and OT treatment). In Oprm1^-/- mice, LIT183 blunted the effects of intranasal OT on the mean duration of nose (H_11,94 = 74.35, p < 0.0001) but not paw contacts (H_11,94 = 74.35, p < 0.0001) and reduced grooming after social contact at 7.5 mg/kg, (H_11,94 = 57.01, p < 0.0001); OT antagonist reduced such grooming in mutant mice treated with vehicle. e We performed a modified version of the 3-chamber test (Oprm1^+/+ vehicle: 6 males – 7 females, Oprm1^+/+ OT: 7 males - 8 females, Oprm1^-/- vehicle: 5 males - 8 females, Oprm1^-/- OT: 6 males – 8 females). During the social preference phase, intranasal OT restored preference for making longer nose contacts with the mouse versus the object in Oprm1 null mice (Genotype x Dose x Stimulus: F_1,47 = 124.01, p < 0.0001), resulting in a fully rescued social preference ratio (H_3,51 = 30.35, p < 0.0001). No effect was detected in Oprm1^+/+ mice. f During the modified social novelty preference phase, OT triggered a preference for making longer nose contacts with the cage mate versus the novel mouse in Oprm1^-/- mice; in contrast, this treatment shifted the preference of Oprm1^+/+ mice towards making longer nose contacts with the novel mouse (G x D x S: F_1,47 = 136.62, p < 0.0001). Similar opposite effects were detected for the social novelty preference ratio (H_3,51 = 37.15, p < 0.0001). Results are shown as scatter plots and mean ± sem. Solid stars: significant difference with the vehicle-treated Oprm1^+/+ group, Tuckey’s post-hoc test following a two-way ANOVA or 2-tailed t-test following a Kruskal-Wallis analysis of variance; open stars: genotype x treatment x stimulus interaction (stimulus: mouse/toy or stranger/cage mate comparison), Tukey’s post-hoc test following a repeated measure analysis of variance (ANOVA); one symbol: p < 0.05, two symbols: p < 0.01; three symbols: p < 0.001. Letters: significant difference with vehicle-treated Oprm1^-/- group (2-tailed t-test or Tukey’s post-hoc test); (c): p < 0.05, (b): p < 0.01, (a): p < 0.001. More behavioural parameters in Fig. S2. C cage mate, IU International Units, M mouse, OT oxytocin, S stranger, SI social interaction, T toy.

Fig. 2. Acute per nasal OT relieved anxiety and induced analgesic effects in Oprm1 null mice but had limited effects on stereotypies and perseveration.

a When administered acutely 5 min before monitoring spontaneous motor stereotypies (Oprm1^+/+ vehicle: 6 males – 6 females, Oprm1^-/- vehicle: 5 males - 5 females, Oprm1^-/- OT 0.15 IU: 4 males – 6 females, other groups: 4 males – 4 females per genotype and dose), per nasal OT increased the number of circling events in Oprm1^+/+ mice. Vehicle-treated Oprm1 null mice displayed more frequent circling behaviour, reduced under OT administration (H_7,72 = 29.78, p < 0.0001). b When exploring the Y-maze (Oprm1^-/- OT 0.3 and 0.6 IU: 4 males – 5 females, other groups: 4 males – 4 females per genotype and dose), vehicle-treated and 0.3 IU OT-treated Oprm1^-/- mice exhibited more frequent same arm returns than Oprm1^+/+ control mice (H_7,66 = 24.1, p < 0.01); this perseverative behaviour was not detected in 0.15 and 0.6 OT-treated mutant mice. c In the novelty-suppressed-feeding test (Oprm1^+/+ and Oprm1^-/- vehicle^: 6 males – 6 females, other groups: 4 males – 4 females per genotype and dose), increased latency to feed in Oprm1^-/- mice was normalized under OT administration (H_7,72 = 39,8, p < 0.0001). d In the tail immersion test (Oprm1^+/+ groups: 4 males – 5 females, Oprm1^-/- vehicle: 4 males – 4 females; Oprm1^-/- OT at 0.15 and 0.5 IU: 5 males ^– 5 females, Oprm1^-/- OT at 0.6 IU: 5 males – 7 females), OT-treated mice (0.6 IU in wild-type mice, all doses in Oprm1 null mice) showed analgesia compared to saline-treated Oprm1^+/+ mice at 48 °C (H_7,76 = 49.4, p < 0.0001). At 50 °C, 0.15 and 0.3 IU of OT produced analgesic effects in Oprm1^-/- mice (H_7,76 = 21.4, p < 0.01). At 52 °C, OT increased nociceptive thresholds only in Oprm1^-/- mice, at doses of 0.15 and 0.3 IU. No significant effect of OT was detected. Results are shown as scatter plots and mean ± sem. Solid stars: significant difference with the vehicle-treated Oprm1^+/+ group, Tuckey’s post-hoc test following a two-way ANOVA or 2-tailed t-test following a Kruskal-Wallis analysis of variance. Letters: significant difference with vehicle-treated Oprm1^-/- group (2-tailed t-test); (c): p < 0.05, (a): p < 0.001. AAR alternate arm returns, NSF novelty-suppressed feeding, SAR same arm returns, SPA spontaneous alternation.

Fig. 3. Chronic intranasal OT maintained prosocial effects in Oprm1 knockout mice while producing a severe social deficit in wild-type controls.

a A first cohort of Oprm1^+/+ and Oprm1^-/- mice was treated daily with either OT (0.3 IU) or vehicle (4 males – 4 females per genotype and treatment) via per nasal route for 17 days. Behavioural testing started on D8. A second cohort received OT (0.3 IU) or vehicle (4 males – 4 females per genotype and treatment) daily for 8 days and was tested for nociception on D8 (blue characters). b In the direct social interaction test, chronic OT restored interaction parameters in Oprm1^-/- mice while it resulted in a severe deficit in Oprm1^+/+ mice (mean duration of nose contacts: G x T: F_1,28 = 666.2, p < 0.0001; mean duration of paw contacts: H_3,32 = 26.8, p < 0.0001, number of following episodes: H_3,32 = 24.3, p < 0.0001, grooming after social contact: H_3,32 = 25.5, p < 0.0001). c Similarly, in the social preference test, repeated OT exposure compromised preference for the mouse over the toy in Oprm1^+/+ mice, but rescued this preference in Oprm1^-/- mice (mean duration of nose contacts: Genotype x Treatment x Stimulus: F_1,28 = 789.8, p < 0.0001, preference ratio: G x T: F_1,28 = 252.1, p < 0.0001). d Oprm1^-/- mice display more frequent circling behaviour, and OT administration had no influence on this stereotyped behaviour (H_3,32 = 13.2, p < 0.01); no effect was detected in Oprm1^+/+ controls. e In the Y-maze, chronic OT failed to suppress perseverative same arm entries (SAR) in Oprm1 mutants, and impaired spontaneous alternation (SPA) in Oprm1^+/+ mice (G x T: F_1,28 = 17.8, p < 0.001). f In the novelty-suppressed feeding test, OT failed to relieve increased latency to eat in Oprm1^-/- mice (H_3,32 = 13.1, p < 0.01). g In the tail immersion test, OT normalized nociceptive thresholds in Oprm1^-/- mice at 48 °C, while inducing analgesia in Oprm1^+/+ controls (G x T: F_1,28 = 10.3, p < 0.01). At 50^°C, chronic OT normalised nociceptive thresholds in Oprm1^-/- mice, without effects in WT mice (G x T: F_1,28 = 50.2, p < 0.0001). Results are shown as scatter plots and mean^±sem. Solid stars: significant difference with the vehicle-treated Oprm1^+/+ group, Tuckey’s post-hoc test following a two-way ANOVA or 2-tailed t-test following a Kruskal-Wallis analysis of variance; open stars: genotype x treatment (Y-maze) or genotype x treatment x stimulus interaction (Social preference - stimulus: mouse/toy), Tukey’s post-hoc test following an analysis of variance (ANOVA); one symbol: p < 0.05, two symbols: p < 0.01; three symbols: p < 0.001. Letters: significant difference with vehicle-treated Oprm1^-/- group (2-tailed t-test or Tukey’s post-hoc test); (c): p < 0.05, (b): p < 0.01, (a): p < 0.001. More behavioural parameters in Fig. S3. 3-Ch: 3-chamber social preference test, AAR alternate arm returns, D day, MB marble burying, MS motor stereotypies, Noci nociception, NSF novelty-suppressed feeding, SAR same arm returns, SPA spontaneous alternation, Y-M Y-maze.

Fig. 4. Prosocial effects of repeated intranasal OT on social deficit in Oprm1 null mice were greater and lasted longer when associated with social experience.

a After a pre-conditioning social interaction session, mice received per nasal OT (0.3 IU) or vehicle administration paired with the presentation of an unfamiliar object (“object” condition) or mouse (“social” condition) every two/three days over 2 weeks (D4 to D15) (4 males – 4 females per genotype, treatment, and conditioning paradigm). A first post-conditioning social interaction session took place on D18, two days before 3-chamber test for social novelty preference (D20). Social interaction was assessed during two additional post-conditioning sessions, a week (D25) and two weeks (D32) after the first post-conditioning session. b During the first post-conditioning social interaction session, OT-treated Oprm1^+/+ mice displayed significant deficits in social behaviour. In contrast, OT improved social behaviour in Oprm1^-/- mice (mean duration of nose contacts: H_7,64 = 50.5, p < 0.0001), more efficiently in mice tested under the “social” paradigm (mean duration of paw contacts: H_7,64 = 37.4, p < 0.0001; grooming after social contact: H_7,64 = 27.3, p < 0.001). c After a week, impaired social interaction was still detected in Oprm1^+/+ mice; among OT-treated Oprm1^-/- mice, only those tested under the “social” paradigm displayed a restoration of social behaviour (mean duration of nose contacts: H_7,64 = 54.6, p < 0.0001; mean duration of paw contacts: H_7,64 = 44.9, p < 0.0001; grooming after social contact: H_7,64 = 36.06, p < 0.0001). d After another week, while a social behaviour deficit was still observed in OT-treated Oprm1^+/+ mice, some prosocial effects of OT conditioning were maintained for Oprm1^-/- mice when tested under the social paradigm only (mean duration of nose contacts: Genotype x Treatment: F_1,56 = 189,3, p < 0.0001; mean duration of paw contacts: H_7,64 = 61.3, p < 0.0001; grooming after social contact: H_7,64 = 44.7, p < 0.0001). e In the three-chamber test, we observed a full restoration of social preference when Oprm1^-/- mice were exposed to OT under the “social” but not “object” setting (mean duration of nose contacts: Stimulus x Treatment x Paradigm: F_1,28 = 27.8, p < 0.0001; preference ratio: H_7,64 = 38.0, p < 0.0001). Results are shown as scatter plots and mean ± sem. Solid stars: significant difference with the vehicle-treated Oprm1^+/+ group, Tuckey’s post-hoc test following a two-way ANOVA or 2-tailed t-test following a Kruskal-Wallis analysis of variance; open stars: genotype x treatment (Y-maze) or genotype x treatment x stimulus interaction (Social preference - stimulus: mouse/toy or stranger/cage mate comparison), Tukey’s post-hoc test following an analysis of variance (ANOVA); daggers: genotype x treatment interaction; one symbol: p < 0.05, two symbols: p < 0.01; three symbols: p < 0.001. Letters: significant difference with vehicle-treated Oprm1^-/- group (2-tailed t-test or Tukey’s post-hoc test); (c): p < 0.05, (b): p < 0.01, (a): p < 0.001. More behavioural parameters in Fig. S4. D day, M mouse, T toy.

Fig. 5. Transcriptional consequences of social OT conditioning in Oprm1 null mice and their wild-type controls.

a In this experiment, OT administration was paired with social encounter for all the mice (“social” paradigm; 4 males – 4 females per genotype and treatment). After a pre-conditioning social interaction session, mice received per nasal OT (0.3 IU) or vehicle administration paired with the presentation of an unfamiliar mouse every two/three days over 2 weeks (D4 to D15). The mice performed a post-conditioning social interaction session on D18 and were sacrificed 45 min after the beginning of behavioural assessment for qRT-PCR analysis. b As observed in the previous experiment, OT exposure had opposite effects on social interaction in Oprm1^+/+ and Oprm1^-/- mice, inducing a severe deficit in the former while rescuing interaction in the latter (mean duration of nose contacts: H_3,32 = 23.3, p < 0.0001; mean duration of paw contacts: H_3,32 = 26.8, p < 0.0001; grooming after social contact: H_3,32 = 25.6, p < 0.0001) (more parameters in Fig. S5). c A hierarchical clustering analysis of qRT-PCR data was performed for each brain region of interest. The most contrasted transcriptional profiles were observed between OT-treated Oprm1^+/+ and OT-treated Oprm1^-/- mice in the NAc, VP/Tu, LS, and CeA, but not in the CPu and MeA where OT exposure led to more similar profiles between Oprm1^+/+ and Oprm1^-/- mice. The main transcriptional effect of OT was to down-regulate gene expression across brain regions (gene names highlighted in green), as seen in the CPu, NAc, VP/Tu, MeA and CeA, but not in the LS. d OT treatment decreased the expression of genes coding for oxytocin and vasopressin receptors (Oxtr, Avpr1a, Avpr1b) in the CPu, VP/Tu and MeA, more significantly in Oprm1^-/- than in Oprm1^+/+ mice. Similarly, OT exposure led a down-regulation of the expression of the main marker genes of SPNs, the genes coding for the dopamine D1 (Drd1a) and D2 (Drd2) receptors, and the gene coding for the adenosine 2a (Adora2) receptor. Such down-regulation was more pronounced in the VP/Tu of the Oprm1^-/- mice. Gene expression data are expressed as fold change versus Oprm1^+/+ - vehicle group (clustering or scatter plots and mean ± SEM). Comparison to Oprm1^+/+ - vehicle group (two-tailed t-test): one star p < 0.05, two stars p < 0.01, three stars p < 0.001. Letters: significant difference with vehicle-treated Oprm1^-/- group (2-tailed t-test); (c): p < 0.05, (b): p < 0.01, (a): p < 0.001. qRT-PCR data used for clustering are displayed in Table S2. More individual transcriptional profiles for candidate genes are displayed in Fig. S5.