Fig. 5: Cholinergic neuronal subtype-specific expression of P11 is involved in specific depression- or anxiety-like phenotypes.
From: Altered acetylcholine modulations and corticoaccumbal pathway in P11-linked social dysfunction
(A, B) Expression of P11-RNA level in cholinergic interneurons (ChAT) of the Nac (A) the brainstem (LDT, B) using RNAscope and IHC in P11-WT mice. (C) Time in the light zone during the entire dark-light box test (0–6 min) for P11flx/flx WT (black) and, ChAT-cP11-KO (blue). Unpaired t-test P = 0.4104. (D) Time in the light zone during the entire dark-light box test with a WT-conspecific introduced in the light area (0–6 min) for P11flx/flx WT (black) and ChAT-cP11-KO (blue). Unpaired t-test P = 0.0002. (E) Time sniffing the WT stimulus during the entire dark-light box test with a WT-conspecific introduced in the light area (0-2 min) for P11flx/flx WT (black) and ChAT-cP11-KO (blue). Unpaired t-test P = 0.001. (F) Time spent sniffing the stress (purple) or neutral (grey) stimulus in the emotional discrimination test for the entire test (0-6 min) for P11flx/flx WT (black) and ChAT-cP11-KO (blue). Paired t-tests: P11-WT 0-2 P = 2.10-6, 2-4 P = 0.0594, 4-6 P = 0.352, 0-6 P = 0.0003. ChAT-cP11-KO: 0-2 P = 0.1675, 2-4 P = 0.9762, 4-6 P = 0.225, 0-6 P = 0.289. P11-WT versus ChAT-cP11-KO: stress P = 1.83.10^6, neutral P = 0.0002. (G) Discrimination index between stress and neutral stimulus during the 1st two min for P11flx/flx WT (black) and ChAT-cP11-KO (blue). Unpaired t-tests to 0.5: P11-WT P = 1.10-9, ChAT-cP11-KO P = 0.0905, P11-WT versus ChAT-cP11-KO P = 0.08916. (H-J) Representative confocal images of the expression of AAV1-2.CBA.RFP.loxP-P11 injected in ChAT-cP11-KO mice targeting the Basal Forebrain (BF, H), LDT (I), or NAC (J). (K) Time in light zone during the entire dark-light box test (0-6 min) for P11-WT mice (injected with AAV, black), ChAT-cP11-KO injected in the BF (grey), the LDT (red) or the NAC (green) or ChAT-cP11-KO (not injected, blue). 1-way ANOVA F(4,33) = 5.06, P = 0.002704, post hoc P11-WT versus AAV-P11-BF: P = 0.0129, P11-WT versus ChAT-cP11-KO: P = 0.0487, ChAT-cP11-KO versus AAV-P11-LDT: P = 0.0187, ChAT-cP11-KO versus AAV-P11-NAC: P = 0.0178. (L) Time in the light zone during the entire dark-light box test with a WT-conspecific introduced in the light area (0-6 min) for P11-WT mice (injected with AAV, black), ChAT-cP11-KO injected in the BF (grey), the LDT (red) or the NAC (green) or ChAT-cP11-KO (not injected, blue). 1-way ANOVA F(4,32) = 6.40, P = 0.000662, Post hoc P11-WT versus ChAT-cP11-KO; P = 0.00016, P11-WT versus AAV-P11-BF: P = 0.03224, P11-WT versus AAV-P11-NAC; P = 0.02331, P11-WT versus AAV-P11-LDT: P = 0.1864; ChAT-cP11-KO versus AAV-P11-LDT: P = 0.6543. (M) Time sniffing the WT stimulus during the entire dark-light box test with a WT-conspecific introduced in the light area (0-6 min) for P11-WT mice (injected with AAV, black), ChAT-cP11-KO injected in the BF (grey), the LDT (red) or the NAC (green) or ChAT-cP11-KO (not injected, blue). 1-way ANOVA F(4,33) = 3.43, P = 0.0187, post hoc ChAT-cP11-KO versus P11-WT; P = 0.00802; AAV-P11-LDT versus ChAT-cP11-KO: P = 0.0452; P11-WT versus AAV-P11-BF: P = 0.01961, AAV-P11-NAC versus P11-WT; P = 0.22734, P11-WT versus AAV-P11-LDT; P = 0.7863, ChAT-cP11-KO versus AAV-P11-BF: P = 0.7269, ChAT-cP11-KO versus AAV-P11-NAC P = 0.11498. (N) Time spent sniffing the stress (purple) or neutral (grey) stimulus in the emotional discrimination test for each segment of 2 min (0-2, 2-4, and 4-6 min) for P11-WT mice (injected with AAV, black), ChAT-cP11-KO injected in the BF (grey), the LDT (red) or the NAC (green). paired t-tests P11-WT: 0-2 min P = 0.0239, 2-4 min P = 0.9818, 4-6 min P = 0.7979, 0-6 min P = 0.5405; AAV-P11-BF: 0-2 min P = 0.047, 2-4 min P = 0.027, 4-6 min P = 0.030, 0-6 min P = 0.000553; AAV-P11-LDT: 0-2 min P = 0.0101, 2-4 min P = 0.056, 4-6 min P = 0.68, 0-6 min P = 0.135; AAV-P11-NAC: 0-2 min P = 0.44, 2-4 min P = 0.97, 4-6 min P = 0.063, 0-6 min P = 0.164. (O) Discrimination index between stress and neutral stimulus during the 1st two min for P11-WT mice (injected with AAV, black), ChAT-cP11-KO injected in the BF (grey), the LDT (red) or the NAC (green). 1-way ANOVA F(1,20) = 2.086, P = 0.1341. Compared to 0.5: P11-WT P = 0.0025, AAV-P11-BF P = 0.0003, AAV-P11-LDT P = 0.1551, AAV-P11-NAC P = 0.1453. (P) Graphical representation of cannula implantation. (Q) Time in light zone during the entire dark-light box test (0-6 min) for P11-WT mice (black) and P11-KO (red) 30-min following administration of saline (0.5 µl), nicotine (100 ng/0.5 µl), oxo (0.1 µg/0.5 µl) or tacrine (1 µg/0.5 µl). 2-way RM-ANOVA treatment effect F(3,33) = 0.117, P = 0.9493, genotype effect F(1,33) = 10.22, P = 0.0085, interaction effect F(3,33) = 0.05, P = 0.983. Post hoc nicotine P11-WT versus P11-KO P = 0.0374. (R) Time in the light zone during the entire dark-light box test with a WT-conspecific introduced in the light area. 2-way RM-ANOVA treatment effect F(3,33) = 9.77, P < 0.0001, genotype effect F(1,33) = 13.88, P = 0.0033, interaction effect F(3,33) = 2.92, P = 0.078. Post hoc saline P11-WT versus P11-KO P = 0.0001, nicotine P11-WT versus P11-KO P = 0.8731, oxo P11-WT versus P11-KO P = 0.1605, tac P11-WT versus P11-KO P = 0.0331. P11-WT saline versus oxo P = 0.0089. P11-KO saline versus nicotine P = 0.0001. (S) Time spent in the stress (purple) or neutral (grey) stimulus zone in the emotional discrimination test for the entire period 90-6 min). Paired t-tests: saline P11-WT P = 0.0129, P11-KO P = 0.69; nicotine P11-WT P = 0.0086 P11-KO P = 0.1317; Oxo P11-WT P = 0.48 P11-KO P = 0.58; tac P11-WT P = 0.088 P11-KO P = 0.0871. P11-WT saline versus nicotine P = 0.0303, P11-WT saline versus oxo P = 0.0193, P11-KO saline versus tac P = 0.0488 (T) Discrimination index between stress and neutral stimulus during the entire 6-min P11-WT mice (black) and P11-KO (red) with administration of cholinergic compound. 2-way RM-ANOVA treatment effect F(3,33) = 0.036, P = 0.102, genotype effect F(1,33) = 0.024, P = 0.102, interaction effect F(3,33) = 0.55, P = 0.8467. Compared to 0.5: P11-WT Saline P = 0.0021, P11-WT nicotine P = 0.0054. Data are represented as mean ± SEM. Individual data are represented as dots when possible. Lines between histograms are used to represent time in a zone or interaction between conditions. *P < 0.05. $ P < 0.05 in comparison with 0.5 DI.
