Oxytocin treatment rescues irritability-like behavior in Cc2d1a conditional knockout mice

Abstract

Irritability, a state of excessive reactivity to negative emotional stimuli, is common in individuals with autism spectrum disorder (ASD). Although it has a significant negative impact of patients' disease severity and quality of life, the neural mechanisms underlying irritability in ASD remain largely unclear. We have previously demonstrated that male mice lacking the Coiled-coil and C2 domain containing 1a (Cc2d1a) in forebrain excitatory neurons recapitulate numerous ASD-like behavioral phenotypes, including impaired social behaviors and pronounced repetitive behaviors. Here, using the bottle-brush test (BBT) to trigger and evaluate aggressive and defensive responses, we show that Cc2d1a deletion increases irritability-like behavior in male but not female mice, which is correlated with reduced number of oxytocin (OXT)-expressing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Intranasal OXT administration or chemogenetic activation of OXT neurons in the PVN rescues irritability-like behavior in Cc2d1a conditional knockout (cKO) mice. Administration of a selective melanocortin receptor 4 agonist, RO27-3225, which potentiates endogenous OXT release, also alleviates irritability-like behavior in Cc2d1a cKO mice, an effect blocked by a specific OXT receptor antagonist, L-368,899. We additionally identify a projection connecting the posterior ventral segment of the medial amygdala (MeApv) and ventromedial nucleus of the ventromedial hypothalamus (VMHvl) for governing irritability-like behavior during the BBT. Chemogenetic suppression of the MeApv-VMHvl pathway alleviates irritability-like behavior in Cc2d1a cKO mice. Together, our study uncovers dysregulation of OXT system in irritability-like behavior in Cc2d1a cKO mice during the BBT and provide translatable insights into the development of OXT-based therapeutics for clinical interventions.

Fig. 1. Male Cc2d1a cKO mice exhibit increased irritability-like behavior in the BBT.

a Schematic showing the experimental schedule for the BBT to trigger and assess irritability-like behavior (aggressive and defensive responses). b Comparison of time spent by male and female WT and Cc2d1a cKO mice in aggressive responses to the BBT (mouse number: N_wt/male = 16, N_cKO/male = 13, N_wt/female = 10, N_cKO/female = 9; two-way ANOVA, genotype: F_(1,44) = 5.13, p = 0.029; sex: F_(1,44) = 19.31, p < 0.0001; interaction: F_(1,44) = 7_.78, p = 0.0078). c Comparison of time spent by male and female WT and Cc2d1a cKO mice in defensive responses to the BBT (mouse number: N_wt/male = 16, N_cKO/male = 13, N_wt/female = 10, N_cKO/female = 9; two-way ANOVA, genotype: F_(1,44) = 14.05, p = 0.0005; sex: F_(1,44) = 1_.27, p = 0.2659; interaction: F_(1,44) = 0_.0₁3, p = 0.9117). d Comparison of time spent by male WT and Cc2d1a cKO mice in individual aggressive responses to the BBT (mouse number: N_wt = 16, N_cKO = 13; two-tailed unpaired Student’s t-test). e Comparison of time spent by male WT and Cc2d1a cKO mice in individual defensive responses to the BBT (mouse number: N_wt = 16, N_cKO = 13; two-tailed unpaired Student’s t-test). f Comparison of time spent by female WT and Cc2d1a cKO mice in individual aggressive responses to the BBT (mouse number: N_wt = 10, N_cKO = 9; two-tailed unpaired Student’s t-test). g Comparison of time spent by female WT and Cc2d1a cKO mice in individual defensive responses to the BBT (mouse number: N_wt = 10, N_cKO = 9; two-tailed unpaired Student’s t-test). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with as compared with each other group.

Fig. 2. Male Cc2d1a cKO mice show reduced number of OXT-expressing neurons in the PVN.

Representative images of OXT immunoreactivity in different anteroposterior levels of the PVN (a) and SON (b) in male WT and Cc2d1a cKO mice. Scale bar, 200 μm. Quantification of percentage of OXT⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 3.35, p = 0.0052; c) and total number of DAPI⁺ cells (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 0.52, p = 0.61; d) in the PVN of male WT and Cc2d1a cKO mice. Quantification of the percentage of OXT⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 0.93, p = 0.48; e) and total number of DAPI⁺ cells (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 0.49, p = 0.63; f) in the SON of male WT and Cc2d1a cKO mice. g Representative images of colocalization of OXT immunoreactivity and P2X4R immunoreactivity in the PVN of male WT and Cc2d1a cKO mice. Scale bar, 200 μm. h Representative images of colocalization of AVP immunoreactivity and P2X4R immunoreactivity in the PVN of male WT and Cc2d1a cKO mice. Scale bar, 200 μm. Quantification of percentage of OXT⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 2.74, p = 0.017; i), OXT⁺P2X4R⁺/P2X4R⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 1.15, p = 0.27; j), P2X4R⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₁₎ = 2.65, p = 0.0224; m) and total number of DAPI⁺ cells (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₁₎ = 1.15, p = 0.2736; n) in the PVN of male WT and Cc2d1a cKO mice. Quantification of the percentage of AVP⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 0.085, p = 0.93; k), AVP⁺P2X4R⁺/P2X4R⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 2.73, p = 0.02; l), P2X4R⁺/DAPI⁺ neurons (mouse number: N_wt = 8, N_cKO = 7; two-tailed paired Student’s t-test, t₍₁₃₎ = 2.29, p = 0.0396; o) and total number of DAPI⁺ cells (mouse number: N_wt = 8, N_cKO = 7; two-tailed unpaired Student’s t-test, t₍₁₃₎ = 0.27, p = 0.7907; p) in the PVN of male WT and Cc2d1a cKO mice. Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01 as compared with WT group.

Fig. 3. Intranasal administration of OXT improves irritability-like behavior in male Cc2d1a cKO mice.

a Schematic representation of the experimental design. Two weeks after stereotaxic injection of AAV₅-pU6-scramble-EGFP or AAV₅-pU6-shmOxt-EGFP into the PVN, mice were single housed for 3 days before the BBT. b Representative image showing the expression of oxytocin and GFP in PVN of shmOxt- and scramble-treated male WT mice. Scale bar: 100 μm. Magnified images of rectangle indicate OXT⁺GFP⁺ cells. Scale bar, 20 µm. c Quantification of the percentage of GFP⁺/DAPI⁺ cells (mouse number: N_scramble = 10, N_shmOxt = 10; two-tailed unpaired Student’s t-test, t₍₁₈₎ = 0.197, p = 0.8454). d Quantification of the percentage of OXT⁺GFP⁺/GFP⁺ cells (mouse number: N_scramble = 10, N_shmOxt = 10; two-tailed unpaired Student’s t-test, t₍₁₈₎ = 20.66, p < 0.0001). e Summary bar graphs depicting the effects of shmOxt and scramble treatment on time spent by male WT in aggressive responses to the BBT (mouse number: N_scramble = 10, N_shmOxt = 10; two-tailed unpaired Student’s t-test, t₍₁₈₎ = 3.572, p = 0.0022). f Summary bar graphs depicting the effects of shmOXT and scramble treatment on time spent by male WT in defensive responses to the BBT (mouse number: N_scramble = 10, N_shmOxt = 10; two-tailed unpaired Student’s t-test, t₍₁₈₎ = 3.114, p = 0.006). g Schematic representation of the experimental design. Male WT or Cc2d1a cKO mice were single housed for 3 days before the BBT. Mice were intranasal administration of OXT (0.2 mg/kg in a volume of 5 μl) or an equivalent volume of vehicle (saline) 30 min before the BBT, respectively. h Summary bar graphs depicting the effect of vehicle and OXT treatment on time spent by male WT or Cc2d1a cKO mice in aggressive responses to the BBT (mouse number: N_wt/Vehicle = 15, N_cKO/Vehicle = 9, N_wt/OXT = 15, N_cKO/OXT = 9; two-way ANOVA, genotype: F_(1,44) = 18_.47, p < 0.0001; treatment: F_(1,44) = 23.69, p < 0_.0001; interaction: F_(1,44) = 15.09, p = 0.0003). i Summary bar graphs depicting the effect of vehicle and OXT treatment on time spent by male WT or Cc2d1a cKO mice in defensive responses to the BBT (mouse number: N_wt/Vehicle = 15, N_cKO/Vehicle = 9, N_wt/OXT = 15, N_cKO/OXT = 9; two-way ANOVA, genotype: F_(1,44) = 11.30, p = 0.0016; treatment: F_(1,44) = 6.11, p = 0.0174; interaction: F_(1,44) = 1.88, p = 0.1778). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with each other group.

Fig. 4. Chemogenetic activation of OXT-expressing neurons in the PVN rescues irritability-like behavior in male Cc2d1a cKO mice.

a Schematic representation of the experimental design. Three weeks after stereotaxic injection of AAV_DJ-mOXT-hM3D(Gq)-mCherry into the PVN, mice were single housed for 3 days before the BBT. Mice were injected intraperitoneally with vehicle (0.5% DMSO in saline) or CNO (3 mg/kg) 30 min before the BBT, respectively. Representative images and bar graphs showing percentage of colocalization of OXT⁺mCherry⁺/OXT⁺ and OXT⁺mCherry⁺/mCherry⁺ neurons in the PVN of male WT (b) and Cc2d1a cKO mice (c). Scale bar, 100 μm. d Summary bar graphs depicting the effects of systemic vehicle and CNO injections on time spent by male WT or Cc2d1a cKO mice that received bilateral intra-PVN AAV_DJ-mOXT-hM3D(Gq)-mCherry injections in aggressive responses to the BBT (mouse number: N_wt/Vehicle = 18, N_cKO/Vehicle = 13, N_wt/CNO = 18, N_cKO/CNO = 13; two-way ANOVA, genotype: F_(1,58) = 30.78, p < 0.0001; treatment: F_(1,58) = 40_.38, p < 0.0001; interaction: F_(1,58) = 35.37, p < 0.0001). e Summary bar graphs depicting the effects of systemic vehicle and CNO injections on time spent by male WT or Cc2d1a cKO mice that received bilateral intra-PVN AAV_DJ-mOXT-hM3D(Gq)-mCherry injections in defensive responses to the BBT (mouse number: N_wt/Vehicle = 18, N_cKO/Vehicle = 13, N_wt/CNO = 18, N_cKO/CNO = 13; two-way ANOVA, genotype: F_(1,58) = 56.16, p < 0.0001; treatment: F_(1,58) = 4.30, p = 0.0426; interaction: F_(1,58) = 4.05, p = 0.0488). f Schematic representation of the experimental design. Three weeks after stereotaxic injection of AAV_DJ-mOXT-hM3D(Gq)-mCherry into the PVN, mice were single housed for 3 days before the BBT. L-368,899 (10 mg/kg) was injected intraperitoneally 10 min before CNO (3 mg/kg) application and CNO was injected intraperitoneally 30 min before the BBT. Representative images and bar graphs showing percentage of colocalization of OXT⁺mCherry⁺/OXT⁺ and OXT⁺mCherry⁺/mCherry⁺ neurons in the PVN of male WT (g) and Cc2d1a cKO mice (h). Scale bar, 100 μm. i Summary bar graphs depicting the effects of systemic L-368,899 and L-368,899 + CNO injections on time spent by male WT or Cc2d1a cKO mice that received bilateral intra-PVN AAV_DJ-mOXT-hM3D(Gq)-mCherry injections in aggressive responses to the BBT (mouse number: N_wt/L-368,899 = 4, N_{cKO/L-368,899} = 7, N_{wt/L-368,899+CNO} = 4, N_{cKO/L-368,899+CNO} = 7; two-way ANOVA, genotype: F_(1,18) = 7.17, p = 0.0154; treatment: F_(1,18) = 10.66, p = 0.0043; interaction: F_(1,18) = 0.06, p = 0.81). j Summary bar graphs depicting the effects of systemic L-368,899 and L-368,899 + CNO injections on time spent by male WT or Cc2d1a cKO mice that received bilateral intra-PVN AAV_DJ-mOXT-hM3D(Gq)-mCherry injections in defensive responses to the BBT (mouse number: N_wt/L-368,899 = 4, N_{cKO/L-368,899} = 7, N_{wt/L-368,899+CNO} = 4, N_{cKO/L-368,899+CNO} = 7; two-way ANOVA, genotype: F_(1,18) = 0.27, p = 0.61; treatment: F_(1,18) = 0.49, p = 0.4915; interaction: F_(1,18) = 0.56, p = 0.4639). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with each other group.

Fig. 5. The MeApv–VmHvl pathway mediates irritability-like behavior.

a Schematic representation of the experimental design. AAV₅-hSyn-DIO-hM4D(Gi)-mCherry or AAV₅-hSyn-DIO-mCherry was bilaterally injected into the MeApv of male Cc2d1a cKO mice. Three weeks after viral infection, mice were individually housed for 3 days and then subjected to the BBT 30 min after vehicle (0.5% DMSO in saline) or CNO injection (3 mg/kg). b Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Cc2d1a cKO mice that received bilateral intra-MeApv injections of either AAV₅-hSyn-DIO-hM4D(Gi)-mCherry or AAV₅-hSyn-DIO-mCherry in aggressive responses to the BBT (mouse number: N_{mCherry/Vehicle} = 4, N_{hM4D(Gi)/Vehicle} = 10, N_mCherry/CNO = 4, N_hM4D(Gi)/CNO = 10; two-way ANOVA, vector: F_(1,24) = 3.44, p = 0.0762; treatment: F_(1,24) = 20.05, p = 0.0002; interaction: F_(1,24) = 11.78, p = 0.0022). c Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Cc2d1a cKO mice that received bilateral intra-MeApv injections of either AAV₅-hSyn-DIO-hM4D(Gi)-mCherry or AAV₅-hSyn-DIO-mCherry in defensive responses to the BBT (mouse number: N_{mCherry/Vehicle} = 4, N_{hM4D(Gi)/Vehicle} = 10, N_mCherry/CNO = 4, N_hM4D(Gi)/CNO = 10; two-way ANOVA, vector: F_(1,24) = 2.67, p = 0.1142; treatment: F_(1,24) = 1.48, p = 0.2352; interaction: F_(1,24) = 3.76, p = 0.0642). d Representative traces showing responses of infected (hM4D(Gi⁾⁺) neurons to 500 ms depolarizing current pulse under whole-cell current clamp before and after bath application of CNO (50 μm) in the ex vivo MeApv slices. e Representative image showing the co-expression of hM4D(Gi)-mCherry and biocytin in MeApv of cKO mice. Scale bar: 25 μm. f Schematic representation of the experimental design. AAV₅-hSyn-DIO-hM3D(Gq)-mCherry or AAV₅-hSyn-DIO-mCherry was bilaterally injected into the MeApv of male Emx1-Cre mice. Three weeks after viral infection, mice were individually housed for 3 days and then subjected to the BBT 30 min after vehicle or CNO injection. g Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Emx1-Cre that received bilateral intra-MeApv injections of either AAV₅-hSyn-DIO-hM3D(Gq)-mCherry or AAV₅-hSyn-DIO-mCherry in aggressive responses to the BBT (mouse number: N_{mCherry/Vehicle} = 6, N_{hM3D(Gq)/Vehicle} = 11, N_mCherry/CNO = 6, N_hM3D(Gq)/CNO = 11; two-way ANOVA, vector: F_(1,30) = 9.41, p = 0.0045; treatment: F_(1,30) = 7.05, p = 0.0126; interaction: F_(1,30) = 11.93, p = 0.0017). h Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Emx1-Cre that received bilateral intra-MeApv injections of either AAV₅-hSyn-DIO-hM3D(Gq)-mCherry or AAV₅-hSyn-DIO-mCherry in defensive responses to the BBT(mouse number: N_{mCherry/Vehicle} = 6, N_{hM3D(Gq)/Vehicle} = 11, N_mCherry/CNO = 6, N_hM3D(Gq)/CNO = 11; two-way ANOVA, vector: F_(1,30) = 4.30, p = 0.0046; treatment: F_(1,30) = 6.85, p = 0.0138; interaction: F_(1,30) = 2.27, p = 0.1424). i Representative traces showing responses of infected (hM3D(Gq)⁺) neurons to 500 ms depolarizing current pulse under whole-cell current clamp before and after bath application of CNO (50 μm) in the ex vivo MeApv slices. j Representative image showing the co-expression of hM3D(Gq)-mCherry and biocytin in MeApv of Emx1-Cre mice. Scale bar: 25 μm. k Schematic representation of the experimental design. AAV_rg-hSyn-DIO-hM4D(Gi)-mCherry or AAV_rg-hSyn-DIO-mCherry was bilaterally injected into the VmHvl of male Cc2d1a cKO mice and CNO (4 mM, 0.5 μl) was micro-infused into the MeApv. l Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Cc2d1a cKO mice that received bilateral intra-VmHvl injections of either AAV_rg-hSyn-DIO-hM4D(Gi)-mCherry or AAV_rg-hSyn-DIO-mCherry in aggressive responses to the BBT (mouse number: N_{mCherry/Vehicle} = 4, N_{hM3D(Gq)/Vehicle} = 7, N_mCherry/CNO = 4, N_hM3D(Gq)/CNO = 7; two-way ANOVA, Vector: F_(1,18) = 7.27, p = 0.0147; Treatment: F_(1,18) = 9.15, p = 0.0073; Interaction: F_(1,18) = 4.10, p = 0.058). m Summary bar graphs depicting the effects of vehicle and CNO treatment on time spent by male Cc2d1a cKO mice that received bilateral intra-VmHvl injections of either AAV_rg-hSyn-DIO-hM4D(Gi)-mCherry or AAV_rg-hSyn-DIO-mCherry in defensive responses to the BBT (mouse number: N_{mCherry/Vehicle} = 4, N_{hM3D(Gq)/Vehicle} = 7, N_mCherry/CNO = 4, N_hM3D(Gq)/CNO = 7; two-way ANOVA, vector: F_(1,18) = 1.88, p = 0.1872; treatment: F_(1,18) = 4.70, p = 0.0438; interaction: F_(1,18) = 11.16, p = 0.0036). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with each other group.