Figure 5. Circuit mechanisms underlying the mania-like behaviors mediated by VTA^Vgat neurons.

(a) Locomotion speed and distance travelled of VTA^Vgat-hM4Di mice that received injections with vehicle or a dopamine receptor antagonist mixture (SCH-23390 and raclopride for D1 and D2/D3 receptors, respectively) and either saline (n= 7 mice) or CNO (n=7 mice). Two-way ANOVA and Bonferroni-Holm post hoc test. F_{(saline or CNO)}=13.6; F_{(vehicle or antagonists)}=91.2; F_{(interaction)}=7.4; Vehicle + saline vs. antagonists + saline t(12)=5, ***p=0.0002; Vehicle + CNO vs. antagonists + CNO t(12)=8.34, ****p=2E-6. All error bars represent the SEM.

(b) Locomotion speed and distance travelled of VTA^Vgat-mCherry and VTA^Vgat-CASP3 mice that received injections with either vehicle (n=6 mice for mCherry and n=10 mice for CASP3) or a dopamine receptor antagonist mixture (n=6 mice for mCherry and n=13 mice for CASP3). Two-way ANOVA and Bonferroni-Holm post hoc test. F_{(mCherry or CASP3)}=10.8; F_{(saline or antagonists)}=4.5; F_{(interaction)}=0.03; mCherry + vehicle vs. mCherry + antagonists t(10)=4.14, **p=0.002; CASP3 + vehicle vs. CASP3 + antagonists t(21)=1.64, p=0.11. All error bars represent the SEM.

(c) Time spent immobile for the tail suspension assay of VTA^Vgat-hM4Di mice that received injections of either vehicle or the dopamine receptor antagonist mixture and saline or CNO. Two-way ANOVA and Bonferroni-Holm post hoc test. F_{(saline or CNO)}=36; F_{(Vehicle or antagonists)}=27; F_{(interaction)}=0.35; vehicle + saline vs. antagonists + saline t(12)=-7.59, ****p=6E-6; vehicle + CNO vs. antagonists + CNO t(12)=-2.54, *p=0.02; antagonists + saline vs. antagonists + CNO t(12)=3.8, **p=0.002. All error bars represent the SEM.

(d) Time spent immobile for the tail suspension assay of VTA^Vgat-mCherry and VTA^Vgat-CASP3 mice that received injections of either vehicle (n=8 mice for mCherry and n=9 mice for CASP3) or the dopamine receptor antagonist mixture (n=8 mice for mCherry and n=9 mice for CASP3). Two-way ANOVA and Bonferroni-Holm post hoc test. F_{(mCherry or CASP3)}=209; F_{(saline or antagonists)}=25; F_{(interaction)}=4.38; mCherry + vehicle vs. mCherry + antagonists t(14)=-4.87, ***p=0.0002; CASP3 + vehicle vs. CASP3 + antagonists t(16)=-2.16, *p=0.04; mCherry + antagonists vs. CASP3 + antagonists t(15)=13, ****p=7e^-10. All error bars represent the SEM.

(e) AAV-DIO-NpHR-mCherry was injected the VTA of Vgat-ires-cre mice, and optic fibers were bilaterally implanted above the LH. The NpHR-mCherry fibers projecting from the VTA^Vgat neurons into the LH were stained by immunohistochemistry (mCherry, red). Scale bar: 200 μm.

(f) Video-tracked paths and distance travelled in the open field by VTA^Vgat-NpHR mice (n=7 mice) with and without opto-inhibition of VTA^Vgat terminals in the LH over 5 minute period. Paired t-test, t(6)=-4.08, **p=0.006.

(g) Time spent immobile on the tail suspension test (TST) of VTA^Vgat-NpHR mice (n=7 mice) with and without opto-inhibition of VTA^Vgat terminals in the LH. Paired t-test, t(6)=5.3, **p=0.001.

(h) Time spent immobile on the forced swimming test (FST) of VTA^Vgat-NpHR mice (n=6 mice) with and without opto-inhibition of VTA^Vgat terminals in the LH. Paired t-test, t(5)=6.77, **p=0.001.

(i) Video-tracked paths and distance travelled in the open field by VTA^Vgat-ChR2 mice (n=8 mice) with and without opto-activation of VTA^Vgat terminals in the LH over 5-minute period. Paired t-test, t(7)=0.18, p=0.85.

(j) Time spent immobile on the tail suspension test (TST) of VTA^Vgat-ChR2 mice (n=8 mice) with and without opto-activation of VTA^Vgat terminals in the LH. Paired t-test, t(7)=-3.11, *p=0.01.

(k) Time spent immobile on the forced swimming test (FST) of VTA^Vgat-ChR2 mice (n=6 mice) with and without opto-activation of VTA^Vgat terminals in the LH. Paired t-test, t(5)=-4, *p=0.01.

(l) Conceptual circuit diagram illustrating VTA^Vgat neurons inhibiting VTA dopamine (DA) neurons and circuitry in the LH. When the VTA^Vgat cells have acutely diminished or absent function, activity of VTA dopamine neurons and arousal-promoting neurons in the LH increase. Valproate can reverse the effects of these changes.