Oxytocin signaling in the ventral tegmental area mediates social isolation-induced craving for social interaction

Abstract

Background: Social interaction is crucial for mental health across animal species. Social experiences, especially in early-life stages, strongly influence brain function and social behavior later in life. Acute social isolation (SI) increases motivation to seek social interaction, but little is known about its underlying neuronal and circuitry mechanisms. Here, we focus on oxytocin signaling in the ventral tegmental area (VTA), a vital node of the brain's reward network, as a potential mechanism for SI-induced craving for social interaction.

Methods: Adolescent (4-week-old) or adult (14-week-old) male C57BL/6J mice underwent a 1-week SI. Free interaction, object exploration, three-chamber social approach, and habituation tests were used to assess social and non-social behavior changes. Viral vectors were used to decipher the underlying neural circuitry, and chemogenetic techniques were applied to modify neuronal activity.

Results: We found that in male C57BL/6J mice, SI during adolescence, but not adulthood, leads to increased craving for social interaction and object exploration, accompanied by impaired social habituation, social novelty preference, and social recognition memory (SRM). SI-induced craving for social interaction and SRM deficit is still observed upon regrouping. Through cell-type-specific manipulations with designer receptors exclusively activated by designer drugs (DREADD), we show that oxytocin neurons in the paraventricular nucleus of the hypothalamus (PVN) are crucial for SI-induced social behavior changes. Chemogenetic activation of PVN oxytocin neurons recapitulates social behavior changes observed in SI mice, whereas chemogenetic inhibition of oxytocin neurons prevents social behavior changes caused by SI. Moreover, we found that dopaminergic neurons in the VTA mediate SI-induced craving for social interaction through their projections to the medial prefrontal cortex (mPFC), but not to the nucleus accumbens. Injection of a specific oxytocin receptor antagonist L368,899 into the VTA or chemical lesions of dopaminergic axon terminals in the mPFC with local application of 6-hydroxydopamine ameliorates SI-induced social behavior changes.

Conclusions: These findings suggest that adolescent SI has enduring effects on social behaviors in male mice through an oxytocinergic modulation of the VTA-to-mPFC dopaminergic circuit activity.

Keywords: Dopamine; Medial prefrontal cortex; Oxytocin; Social craving; Social isolation; Ventral tegmental area.

Fig. 1

Adolescent SI induces social behavioral changes in male mice. A Schematic diagram illustrating the experimental pipeline. After weaning (P21), male mice were housed in groups of the same sex until P27 and housed either in groups or alone between P28 and P34. A series of behavioral tests consisting of the free interaction test, object exploration, 3-chamber test, and habituation test were conducted between P35 and P38. B Top, schematic representation of the free interaction test. Bottom, bar graphs with dots showing the time spent by the male subject mouse in social interaction with a novel mouse. SI mice spend more time interacting with novel mice compared with GH mice [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 14.53, P < 0.0001, 95% CI (99.32–132.7)]. C Top, schematic representation of the object exploration test. Bottom, bar graphs with dots showing the time spent by the subject mouse exploring the novel object. SI mice spend more time exploring the novel object compared with GH mice [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.94, P = 0.0083, 95% CI (3.77–22.31)]. D Top, schematic representation of the habituation test. Bottom, scatterplots showing the time spent by the subject mouse in social interaction with the same stimulus mouse for 4 consecutive 5-min trials with an inter-trial interval of 10 min. SI mice exhibited a significant habituation deficit compared with GH mice [mouse number: GH: n = 10; SI: n = 11; two-way RM ANOVA, trial: F_{(2.612,49.63)} = 14.13, P < 0.0001; housing condition: F_(1,19) = 9.47, P = 0.0062; trial × housing condition interaction: F_(3,57) = 6.09, P = 0.0011]. E Left, schematic representation of the 3-chamber sociability test. Middle, bar graphs with dots showing the time spent by the male subject mouse in sniffing directed at a wire cage containing the juvenile stimulus mouse (S) or an empty wire cage (E). Both SI and GH subject mice spent significantly more time interacting with the wire cage containing the juvenile stimulus mouse than the empty wire cage. Right, the discrimination index (stimulus minus empty) was similar between SI and GH subject mice in the sociability test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 1.10, P = 0.28, 95% CI (−0.21 to 0.07)]. F Left, schematic representation of the 3-chamber social novelty preference test. Middle, bar graphs with dots showing the time spent by the male subject mouse in sniffing directed at the wire cage containing a familiar mouse (F) or a novel mouse (N1) 10 min after the sociability test. Both SI and GH subject mice spent significantly more time sniffing the cage containing the novel mouse than the familiar mouse. Right, the discrimination index (novel 1 minus familiar) of SI subject mice was significantly less than that of GH subject mice in the social novelty preference test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.11, P = 0.0485, 95% CI (−0.48 to −0.002)]. G Left, schematic representation of the 3-chamber SRM test. Middle, bar graphs with dots showing the time spent by the male subject mouse in sniffing directed at the wire cage containing a familiar mouse (N1) or a novel 2 mouse (N2), 1 day after the initial interaction. GH, but not SI, subject mice spent significantly more time sniffing the cage containing the novel mouse than the familiar mouse (mouse number: GH: n = 10; SI: n = 11; paired Student’s t-test; t₍₁₀₎ = 0.29, P = 0.78, 95% CI (−7.30 to 9.52). Right, the discrimination index (novel 2 minus familiar) of SI subject mice was significantly less than that of GH subject mice in the SRM test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.81, P = 0.011, 95% CI (−0.60 to −0.09)]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. Panels B–G were created with BioRender.com

Fig. 2

Resocialization is insufficient to rescue receptive social behavioral changes by adolescent SI. A Schematic diagram illustrating the experimental pipeline. Male mice were housed either in groups or alone between P28 and P34. SI mice were regrouped (rGH) until P55 and GH mice were always kept in a group. A series of behavioral tests consisting of the free interaction test, object exploration, three-chamber test, and habituation test were performed between P56 and P59. B Top, schematic representation of the free interaction test. Male rGH mice spend more time interacting with the novel mice compared with male GH mice [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.91, P = 0.0089, 95% CI (7.70–46.98)]. C Top, schematic representation of the object exploration test. Bottom, the time spent exploring the novel object was comparable between male rGH and GH mice [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 0.38, P = 0.71, 95% CI (−10.69 to 15.47)]. D Top, schematic representation of the habituation test. Bottom, male rGH and GH mice exhibited similar behavioral habituation to repeated social stimulation [mouse number: GH: n = 10; SI: n = 11; two-way RM ANOVA, trial: F_(3,57) = 16.49, P < 0.0001; housing condition: F_(1,19) = 0.38, P = 0.5472; trial × housing condition interaction: F_(3,57) = 0.52, P = 0.6686]. E Left, schematic representation of the 3-chamber sociability test. Middle, male rGH and GH subject mice spent significantly more time interacting with the wire cage containing the juvenile stimulus mouse (S) than the empty wire cage (E). Right, the discrimination index (stimulus minus empty) was comparable between male rGH and GH subject mice in the sociability test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 0.63, P = 0.536, 95% CI (−0.06 to 0.11)]. F Left, schematic representation of the 3-chamber social novelty preference test. Middle, male rGH and GH subject mice spent significantly more time sniffing the cage containing the novel mouse (N1) than the familiar mouse (F). Right, the discrimination index (novel 1 minus familiar) was comparable between male rGH and GH subject mice in the social novelty preference test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 1.37, P = 0.188, 95% CI (−0.20 to 0.04)]. G Left, schematic representation of the 3-chamber SRM test. Middle, male GH, but not male dGH, subject mice spent significantly more time sniffing the cage containing the novel mouse (N2) than the familiar mouse (N1). Right, the discrimination index (novel 2 minus familiar) of male rGH subject mice was significantly less than male GH subject mice in the SRM test [mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 3.09, P = 0.006, 95% CI (−0.48 to −0.09)]. Data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001. Panels B–G were created with BioRender.com

Fig. 3

PVN oxytocin projections to VTA dopamine neurons mediate SI-induced craving for social interaction. A Schematic representation of the experimental design. AAV_DJ-hSyn-DIO-hM4D(Gi)-mCherry was bilaterally injected into the PVN of male Oxytocin-Ires-Cre mice. One week after viral infection, mice were housed either in groups or alone, treated with CNO or vehicle in their drinking water for 1 week, and then subjected to the free interaction test, object exploration, and habituation test. B Representative images showing the co-expression of hM4D(Gi)-mCherry and oxytocin immunoreactivity in the PVN (left). Scale bar, 100 μm. Right, magnification of the boxed area; scale bar, 20 μm. C Behavioral performance of mice in the free interaction test. There were no significant differences among male vehicle-treated GH, CNO-treated SI, and CNO-treated GH mice in the time spent interacting with a novel mouse [mouse number: GH/vehicle: n = 7; SI/vehicle: n = 7; GH/CNO: n = 9; SI/CNO: n = 8; two-way ANOVA, group: F_(1,27) = 6.183, P = 0.0194; treatment: F_(1,27) = 5.12, P = 0.0319; group × treatment interaction: F_(1,27) = 4.33, P = 0.047]. D Behavioral performance of mice in the object exploration test. There was a significant difference between male vehicle-treated SI and CNO-treated SI mice while exploring the novel object [mouse number: GH/vehicle: n = 7; SI/vehicle: n = 7; GH/CNO: n = 9; SI/CNO: n = 8; two-way ANOVA, group: F_(1,27) = 7.80, P = 0.0095; treatment: F_(1,27) = 4.72, P = 0.0387; group × treatment interaction: F_(1,27) = 7.45, P = 0.011]. E Behavioral performance of mice in the habituation test. There was a significant difference between male vehicle-treated SI and CNO-treated SI mice in behavioral habituation to repeated social stimulation [mouse number: GH/vehicle: n = 7; SI/vehicle: n = 7; GH/CNO: n = 9; SI/CNO: n = 8; two-way RM ANOVA, trial: F_(2.65,71.55) = 63.85, P < 0.0001; treatment: F_(3,27) = 8.56, P = 0.0004; trial × treatment interaction: F_(9,81) = 4.39, P = 0.0001; Tukey’s post hoc multiple comparisons test, P_{GH/Vehicle vs. SI/Vehicle} < 0.0001, P_{GH/Vehicle vs. SI/CNO} = 0.0309, P_{SI/Vehicle vs. GH/CNO} = 0.0002, P_{SI/Vehicle vs. SI/CNO} = 0.0386]. F Schematic representation of the experimental design. AAV_DJ-hSyn-DIO-hM3D(Gq)-mCherry was bilaterally injected into the PVN of male Oxytocin-Ires-Cre mice. One week after viral infection, mice were housed in groups, treated with vehicle or CNO in their drinking water for 1 week, and then subjected to the free interaction test, object exploration, and habituation test. G Representative images showing the co-expression of hM3D(Gq)-mCherry and oxytocin immunoreactivity in the PVN (left). Scale bar, 100 μm. Right, magnification of the boxed area; scale bar, 20 μm. H Behavioral performance of mice in the free interaction test. CNO-treated GH mice spend more time interacting with the novel mice compared with vehicle-treated GH mice [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 7; GH/CNO: n = 8; SI/CNO: n = 9; two-way ANOVA, group: F_(1,28) = 0.91, P = 0.3475; treatment: F_(1,28) = 0.53, P = 0.4734; group × treatment interaction: F_(1,28) = 10.92, P = 0.0026]. I Behavioral performance of mice in the object exploration test. CNO-treated GH mice spend more time exploring the novel object compared with vehicle-treated GH mice [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 7; GH/CNO: n = 8; SI/CNO: n = 9; two-way ANOVA, group: F_(1,28) = 4.22, P = 0.0494; treatment: F_(1,28) = 3.25, P = 0.0823; group × treatment interaction: F_(1,28) = 5.36, P = 0.0281]. J Behavioral performance of mice in the habituation test. CNO-treated GH mice exhibited a significant habituation deficit compared with vehicle-treated GH mice [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 7; GH/CNO: n = 8; SI/CNO: n = 9; two-way RM ANOVA, trial: F_{(2.746,79.62)} = 32.64, P < 0.0001; treatment: F_(3,29) = 4.87, P = 0.0073; trial × treatment interaction: F_(9,87) = 3.45, P = 0.0011; Tukey’s post hoc multiple comparisons test, P_{GH/Vehicle vs. SI/Vehicle} = 0.0007, P_{GH/Vehicle vs. GH/CNO} = 0.0107, P_{GH/Vehicle vs. SI/CNO} = 0.0099]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. Panels A and F were created with BioRender.com

Fig. 4

VTA dopamine neuron activity encodes SI-induced craving for social interaction. A Schematic representation of the experimental design. AAV₉-TH-Cre and AAV₅-hSyn-DIO-GCaMP6s were unilaterally injected into the VTA of male mice and implanted in an optical fiber above the injection site. One week after viral infection, mice were individually housed for 1 week, and then the Ca²⁺ signal of VTA dopamine neurons was recorded at P35 while the subject mouse was engaged in the free social interaction test. B Representative image showing Cre-dependent expression of GCaMP6s in VTA dopamine neurons of a mouse with optic fiber placement indicated. Scale bar, 100 μm. C Representative heat maps (top) and average ΔF/F traces (bottom) of dopamine neuron GCaMP6 signals from one example SI mouse and one example of GH mouse aligned to the onset of investigation of a novel conspecific. The trials were obtained from the same GH mouse and SI mouse. D Bar graphs with dots showing the quantitative analyses of the peak of Z-score [left; mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.33, P = 0.03, 95% CI (0.19 to 3.62)] and the area under the curve [UC; right; mouse number: GH: n = 10; SI: n = 11; two-tailed unpaired Student’s t-test; t₍₁₉₎ = 2.70, P = 0.014, 95% CI (1.66 to 13.20)] of the GCaMP signals when male SI or GH mice interacted with the novel mice in the free interaction test. E Schematic representation of the experimental design. AAV_retro-hSyn-DIO-hM4D(Gi)-mCherry or AAV_retro-hSyn-DIO-mCherry was bilaterally injected into the VTA of male Oxytocin-Ires-Cre mice. One week after viral infection, mice were housed either in groups or alone, treated with CNO in their drinking water for 1 week, and then subjected to the free interaction test, object exploration, and habituation test. F Representative images showing the co-expression of hM4D(Gi)-mCherry and oxytocin immunoreactivity in the PVN (left). Scale bar, 100 μm. Right, magnification of the boxed area; scale bar, 100 μm. G Behavioral performance of mice in the free interaction test. There were no significant differences among male mCherry-treated GH, hM4D(Gi)-treated SI, and hM4D(Gi)-treated GH mice in the time spent interacting with a novel mouse [mouse number: GH/mCherry/CNO: n = 5; SI/mCherry/CNO: n = 5; GH/hM4D(Gi)/CNO: n = 7; SI/hM4D(Gi)/CNO: n = 6; two-way ANOVA, group: F_(1,20) = 12.41, P = 0.0021; treatment: F_(1,20) = 9.88, P = 0.0051; group × treatment interaction: F_(1,20) = 6.33, P = 0.0205]. H Behavioral performance of mice in the object exploration test. There were no significant differences between male mCherry-treated SI and hM4D(Gi)-treated SI mice in the time exploring the novel object [mouse number: GH/mCherry/CNO: n = 5; SI/mCherry/CNO: n = 5; GH/hM4D(Gi)/CNO: n = 7; SI/hM4D(Gi)/CNO: n = 6; two-way ANOVA, group: F_(1,20) = 20.65, P = 0.0002; treatment: F_(1,20) = 0.08, P = 0.787; group × treatment interaction: F_(1,20) = 0.06, P = 0.8025]. I Behavioral performance of mice in the habituation test. There was a significant difference between male mCherry-treated SI and hM4D(Gi)-treated SI mice in behavioral habituation to repeated social stimulation [mouse number: GH/mCherry/CNO: n = 5; SI/mCherry/CNO: n = 5; GH/hM4D(Gi)/CNO: n = 7; SI/hM4D(Gi)/CNO: n = 6; two-way RM ANOVA, trial: F_{(2.398,45.57)} = 53.73, P < 0.0001; treatment: F_(3,19) = 4.09, P = 0.0213; trial × treatment interaction: F_(9,57) = 2.20, P = 0.0355; Tukey’s post hoc multiple comparisons test, P_{GH/mCherry/CNO vs. SI/mCherry/CNO} = 0.005, P_{SI/mCherry/CNO vs. GH/hM4D(Gi)/CNO} = 0.0014, P_{SI/mCherry/CNO vs. SI/hM4D(Gi)/CNO} = 0.0316]. J Schematic representation of the experimental design. Vehicle (saline) or L-368,899 was bilaterally injected into the VTA of male SI or GH mice 20 min before social behavioral tests. K Representative coronal section showing bilateral cannula tracks targeting the VTA. Scale bar: 100 μm. L Behavioral performance of mice in the free interaction test. There was a significant difference between male vehicle-treated SI and L-368,899-treated SI mice in the time spent interacting with a novel mouse [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 8; GH/L-368,899: n = 8; SI/L-368,899: n = 8; two-way ANOVA, group: F_(1,28) = 14.92, P = 0.0006; treatment: F_(1,28) = 5.86, P = 0.0222; group × treatment interaction: F_(1,28) = 4.25, P = 0.0486]. M Behavioral performance of mice in the object exploration test. There was no significant difference between male vehicle-treated SI and L-368,899-treated SI mice in the time exploring the novel object [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 8; GH/L-368,899: n = 8; SI/L-368,899: n = 8; two-way ANOVA, group: F_(1,28) = 19.31, P = 0.0001; treatment: F_(1,28) = 1.00, P = 0.3242; group × treatment interaction: F_(1,28) = 0.0536, P = 0.8186]. N Behavioral performance of mice in the habituation test. There was a significant difference between male vehicle-treated SI and L-368,899-treated SI mice in behavioral habituation to repeated social stimulation [mouse number: GH/vehicle: n = 8; SI/vehicle: n = 8; GH/L-368,899: n = 8; SI/L-368,899: n = 8; two-way RM ANOVA, trial: F_{(2.550,71.41)} = 48.98, P < 0.0001; treatment: F_(3,28) = 11.08, P < 0.0001; trial × treatment interaction: F_(9,84) = 5.67, P < 0.0001; Tukey’s post hoc multiple comparisons test, P_{GH/Vehicle vs. SI/Vehicle} < 0.0001, P_{SI/Vehicle vs. GH/L-368,899} < 0.0001, P_{SI/Vehicle vs. SI/L-368,899} < 0.0001]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 5

Dopamine release in the mPFC is needed for SI-induced craving for social interaction. A Schematic representation of the experimental design. One week after the local injection of vehicle (0.2% ascorbic acid in saline) or 6-hydroxydopamine (6-OHDA) into the mPFC, male mice were housed either in groups or alone for 1 week and then subjected to the free interaction test, object exploration, and habituation test. B Representative images of the mPFC illustrating the loss of tyrosine hydroxylase (TH) labeling following ablation of dopaminergic axon terminals by local injection of 6-OHDA compared with vehicle treatment. Scale bars, 100 μm. C Behavioral performance of mice in the free interaction test. There were no significant differences among male vehicle-treated GH, 6-OHDA-treated GH and 6-OHDA-treated SI mice in the time spent interacting with a novel mouse [mouse number: GH/vehicle: n = 6; SI/vehicle: n = 8; GH/6-OHDA: n = 8; SI/6-OHDA: n = 12; two-way ANOVA, group: F_(1,30) = 48.15, P < 0.0001; treatment: F_(1,30) = 22.58, P < 0.0001; group × treatment interaction: F_(1,30) = 16.37, P = 0.0003]. D Behavioral performance of mice in the object exploration test. There was still a significant increase in the time exploring the novel object in 6-OHDA-treated SI mice compared with vehicle-treated GH mice [mouse number: GH/vehicle: n = 6; SI/vehicle: n = 8; GH/6-OHDA: n = 8; SI/6-OHDA: n = 12; two-way ANOVA, group: F_(1,30) = 19.00, P = 0.0001; treatment: F_(1,30) = 5.560, P = 0.0251; group × treatment interaction: F_(1,30) = 1.064, P = 0.3106]. E Behavioral performance of mice in the habituation test. There was a significant difference between male vehicle-treated SI and 6-OHDA-treated SI mice in behavioral habituation to repeated social stimulation [mouse number: GH/vehicle: n = 6; SI/vehicle: n = 8; GH/6-OHDA: n = 8; SI/6-OHDA: n = 12; two-way RM ANOVA, trial: F_{(2.237,67.10)} = 36.93, P < 0.0001; treatment: F_(3,30) = 10.49, P < 0.0001; trial × treatment interaction: F_(9,90) = 5.853, P < 0.0001; Tukey’s post hoc multiple comparisons test, P_{GH/vehicle vs. SI/vehicle} < 0.0001, P_{SI/vehicle vs. GH/6-OHDA} < 0.0001, P_{SI/vehicle vs. SI/6-OHDA} < 0.0001]. F Schematic representation of the experimental design. One week after the local injection of saline or 6-OHDA into the NAc, male mice were housed either in groups or alone for 1 week and then subjected to the free interaction test, object exploration, and habituation test. G Representative images of the NAc illustrating loss of TH labeling following ablation of dopaminergic axon terminals by local injection of 6-OHDA compared with vehicle treatment. Scale bars, 100 μm. H Behavioral performance of mice in the free interaction test. Male vehicle-treated SI and 6-OHDA-treated SI mice spend more time interacting with the novel mice than male vehicle-treated GH and 6-OHDA-treated GH mice [mouse number: GH/vehicle: n = 14; SI/vehicle: n = 10; GH/6-OHDA: n = 7; SI/6-OHDA: n = 11; two-way ANOVA, group: F_(1,38) = 34.42, P < 0.0001; treatment: F_(1,38) = 1.463, P = 0.2339; group × treatment interaction: F_(1,38) = 2.460, P = 0.1251]. I Behavioral performance of mice in the object exploration test. Male vehicle-treated SI and 6-OHDA-treated SI mice spend more time exploring the novel object than male vehicle-treated GH and 6-OHDA-treated GH mice [mouse number: GH/vehicle: n = 14; SI/vehicle: n = 10; GH/6-OHDA: n = 7; SI/6-OHDA: n = 11; two-way ANOVA, group: F_(1,38) = 24.44, P < 0.0001; treatment: F_(1,38) = 0.1198, P = 0.7312; group × treatment interaction: F_(1,38) = 0.04, P = 0.8341]. J Behavioral performance of mice in the habituation test. Male vehicle-treated SI and 6-OHDA-treated SI mice exhibited significant habituation deficits compared with male vehicle-treated GH and 6-OHDA-treated GH mice [mouse number: GH/vehicle: n = 14; SI/vehicle: n = 10; GH/6-OHDA: n = 7; SI/6-OHDA: n = 11; two-way RM ANOVA, trial: F_{(2.815,107.0)} = 67.53, P < 0.0001; treatment: F_(3,38) = 4.00, P = 0.0143; trial × treatment interaction: F_(9,114) = 3.83, P = 0.0003; Tukey’s post hoc multiple comparisons test, P_{GH/vehicle vs. SI/vehicle} = 0.0047, P_{GH/vehicle vs. SI/6-OHDA} = 0.0016, P_{SI/vehicle vs. GH/6-OHDA} = 0.0284, P_{GH/6-OHDA vs. SI/6-OHDA} = 0.0195]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. Panels A and F were created with BioRender.com

Fig. 6

The dopaminergic pathway from the VTA to the mPFC orchestrates the expression of heightened social interaction in SI mice. A Schematic representation of the experimental design. AAV₉-TH-Cre was bilaterally injected into the VTA and AAV_retro-hSyn-DIO-hM4D(Gi)-mCherry or AAV_retro-hSyn-DIO-mCherry was bilaterally injected into the mPFC of male mice. One week after viral infection, mice were housed either in groups or alone, and then subjected to the free interaction test, object exploration, and habituation test. CNO (3 mg/kg) was administered intraperitoneally 30 min prior to the behavioral test. B Representative images showing the co-expression of hM4D(Gi)-mCherry and TH immunoreactivity in the VTA. Scale bar, 100 μm. C Behavioral performance of mice in the free interaction test. There were no significant differences among male mCherry-treated GH, hM4D(Gi)-treated SI, and hM4D(Gi)-treated GH mice in the time spent interacting with a novel mouse [mouse number: GH/mCherry/CNO: n = 6; SI/mCherry/CNO: n = 6; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 7; two-way ANOVA, group: F_(1,23) = 86.52, P < 0.0001; treatment: F_(1,23) = 53.57, P < 0.0001; group × treatment interaction: F_(1,23) = 42.11, P < 0.0001]. D Behavioral performance of mice in the object exploration test. There was no significant difference between male mCherry-treated SI and hM4D(Gi)-treated SI mice in the time exploring the novel object [mouse number: GH/mCherry/CNO: n = 6; SI/mCherry/CNO: n = 6; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 7; two-way ANOVA, group: F_(1,23) = 16.35, P = 0.0005; treatment: F_(1,23) = 0.2534, P = 0.6195; group × treatment interaction: F_(1,23) = 0.02, P = 0.8967]. E Behavioral performance of mice in the habituation test. There was a significant difference between male mCherry-treated SI and hM4D(Gi)-treated SI mice in behavioral habituation to repeated social stimulation [mouse number: GH/mCherry/CNO: n = 6; SI/mCherry/CNO: n = 5; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 7; two-way RM ANOVA, trial: F_{(2.694,59.28)} = 60.26, P < 0.0001; treatment: F_(3,22) = 11.61, P < 0.0001; trial × treatment interaction: F_(9,66) = 3.475, P = 0.0015; Tukey’s post hoc multiple comparisons test, P_{GH/mCherry/CNO vs. SI/mCherry/CNO} = 0.0002, P_{SI/mCherry/CNO vs. GH/hM4D(Gi)/CNO} < 0.0001, P_{SI/mCherry/CNO vs. SI/hM4D(Gi)/CNO} = 0.0026]. F Schematic representation of the experimental design. AAV₉-TH-Cre was bilaterally injected into the VTA, and AAV_retro-hSyn-DIO-hM4D(Gi)-mCherry or AAV_retro-hSyn-DIO-mCherry was bilaterally injected into the NAc of male mice. One week after viral infection, mice were housed either in groups or alone, and then subjected to the free interaction test, object exploration, and habituation test. CNO was administered intraperitoneally 30 min prior to the behavioral test. G Representative images showing the co-expression of hM4D(Gi)-mCherry and TH immunoreactivity in the VTA. Scale bar, 100 μm. H Behavioral performance of mice in the free interaction test. Male mCherry-treated SI and hM4D(Gi)-treated SI mice spend more time interacting with the novel mice compared with male mCherry-treated GH and hM4D(Gi)-treated GH mice [mouse number: GH/mCherry/CNO: n = 8; SI/mCherry/CNO: n = 7; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 8; two-way ANOVA, group: F_(1,27) = 47.98, P < 0.0001; treatment: F_(1,27) = 0.05, P = 0.8335; group × treatment interaction: F_(1,27) = 0.95, P = 0.3374]. I Behavioral performance of mice in the object exploration test. Male mCherry-treated SI and hM4D(Gi)-treated SI mice spend more time exploring the novel object compared with male mCherry-treated GH and hM4D(Gi)-treated GH mice [mouse number: GH/mCherry/CNO: n = 8; SI/mCherry/CNO: n = 7; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 8; two-way ANOVA, group: F_(1,27) = 44.50, P < 0.0001; treatment: F_(1,27) = 0.0008, P = 0.9773; group × treatment interaction: F_(1,27) = 0.42, P = 0.5222]. J Behavioral performance of mice in the habituation test. Male mCherry-treated SI and hM4D(Gi)-treated SI mice exhibited significant habituation deficits compared with male mCherry-treated GH and hM4D(Gi)-treated GH mice [mouse number: GH/mCherry/CNO: n = 8; SI/mCherry/CNO: n = 7; GH/hM4D(Gi)/CNO: n = 8; SI/hM4D(Gi)/CNO: n = 8; two-way RM ANOVA, trial: F_{(2.430,65.62)} = 77.81, P < 0.0001; treatment: F_(3,27) = 12.43, P < 0.0001; trial × treatment interaction: F_(9,81) = 5.631, P < 0.0001; Tukey’s post hoc multiple comparisons test, P_{GH/mCherry/CNO vs. SI/mCherry/CNO} = 0.0001, P_{GH/mCherry/CNO vs. SI/hM4D(Gi)/CNO} = 0.0002, P_{SI/mCherry/CNO vs. GH/hM4D(Gi)/CNO} = 0.0017, P_{GH/hM4D(Gi)/CNO vs. SI/hM4D(Gi)/CNO} = 0.0036]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. Panels A and F were created with BioRender.com