Fig. 3.
Selective chemogenetic inhibition of LDTg cholinergic but not glutamatergic neurons during CSD is sufficient to prevent depressive behaviors and VTA DA dysregulation. a Bilateral stereotaxic injection of AAV8-hSyn-DIO-hM4-mcherry in the LDTg of ChAT-Cre mice (LDTgChAT-hM4 mice). Confocal images showing colocalization of mcherry and vesicular acetylcholinesterase transporter (vAChT) Scale bar = 20 µm. Epifluorescence micrograph showing cholinergic fibers expressing mcherry in close interaction with tyrosine hydroxylase (TH)-positive VTA dopamine neurons. Scale bar = 25 µm. b Social interaction times in undefeated or defeated LDTgChAT-hM4 mice treated with saline or CNO. Saline-injected defeated mice showed marked social aversion, whereas CNO injection in the defeated group restored social interaction (number of mice: Naive/Sal = 8; Naive/CNO = 8; CSD/Sal = 18; CSD/CNO = 16). Interaction treatment × target F(3,46) = 9.62, P = 0.0001; Interaction treatment × stress F(1,46) = 7.99, P < 0.01; repeated-measures two-way ANOVA followed by Sidak’s comparisons test, asterisks depict NTg vs. Tg comparisons, hash depicts comparisons between saline vs. CNO in the Tg condition; **P < 0.01, ***P < 0.001, ###P < 0.001. c Inhibition of cholinergic LDTg neurons during CSD prevented increased excitability in VTA DA neurons (number of cell/mice: Naive/Sal = 11/4; Naive/CNO = 13/4; CSD/Sal = 22/5; CSD/CNO = 18/5). Interaction treatment × current F(18,360) = 4.66, P = 0.0001; repeated-measures two-way ANOVA followed by Sidak’s comparisons test ***P < 0.001. d Bilateral stereotaxic LDTg injection of AAV8-hSyn-DIO-hM4-mcherry of Vglut2-Cre mice (LDTgVglut2-hM4 mice). Confocal image shows representative injection site. Confocal image showing glutamatergic fibers expressing mcherry in close interaction with TH-positive VTA dopamine neurons. Scale bar = 20 µm. e Social interaction times in undefeated or defeated LDTgVglut2-hM4 mice treated with saline or CNO. Silencing of LDTg glutamatergic neurons by administration of CNO during stress did not prevent the appearance of social aversion (number of mice: Naive/Sal = 8; Naive/CNO = 9; CSD/Sal = 13; CSD/CNO = 12). Interaction treatment × target F(3,38) = 6.80, P = 0.0009; Interaction treatment × stress F(1,38) = 0.1619, P = 0.6897; repeated-measures two-way ANOVA followed by Sidak’s comparisons test, asterisks depict NTg vs. Tg comparisons, hash depicts comparisons between saline vs. CNO in the Tg condition; **P < 0.01, ***P < 0.001, ###P < 0.001. f Patch-clamp recordings in brain slices showed that inhibition of glutamatergic LDTg neurons during CSD did not prevent increased excitability in VTA DA neurons (number of cell/mice per condition: Naive/Sal = 12/4; Naive/CNO = 14/4; CSD/Sal = 10/5; CSD/CNO = 10/5). Interaction treatment × current F(6,108) = 0.95, P = 0.466; repeated-measures two-way ANOVA followed by Sidak’s comparisons test ***P < 0.001). All plots depict mean ± SEM