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. AAVretro-hSyn-DIO-hM4D(Gi)-mCherry or AAVretro-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, PGH/mCherry/CNO vs. SI/mCherry/CNO = 0.005, PSI/mCherry/CNO vs. GH/hM4D(Gi)/CNO = 0.0014, PSI/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, PGH/Vehicle vs. SI/Vehicle < 0.0001, PSI/Vehicle vs. GH/L-368,899 < 0.0001, PSI/Vehicle vs. SI/L-368,899 < 0.0001]. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001