Fig 2: Trait anxiety is associated with a Crhr1 common variant and with different CRHR1 expression in VTA DA neurons.(A to C) All rats (n = 26 HA, n = 32 IA, and n = 35 LA) were genotyped for SNP on rs106600307 located on chr10:92191940. (A) Allele pair distribution is associated with time spent in the open arms in the EPM (Kruskal-Wallis ?2 = 6.2139, df = 2, P = 0.04474). (B) Heterozygous rats spent significantly more time in the open arms of the EPM compared to homozygous rats (Wilcoxon rank sum test, W = 1397.5, P = 0.01309). Bars represent the percentage of time spent in the open arms (%OA) for the different allele pairs. (C) A significantly higher proportion of LA rats than HA rats are heterozygous for the Crhr1 SNP (?2 test6.146,1, P = 0.0132). (D to F) CRHR1 expression in DA cells in the VTA was probed with RNAscope. Expression of CRHR1 mRNA (red) in TH+ cells (green), along with nuclear staining with DAPI (blue) and merged image in the VTA of LA (D) and HA (E) rats. (F) Left: Higher magnification images of the marked area of panels in (D) and (E) in LA and HA rats. Right: LA rats had higher CRHR1 mRNA expression in DA neurons in the VTA than HA rats (Mann-Whitney test, U = 2.000, P = 0.032). (G) Left: CRHR1 expression in GABAergic cells in the VTA. Merged images of expression of CRHR1 mRNA (red) in GAD1+ cells (magenta), along with nuclear staining with DAPI (blue) in LA and HA rats. Right: CRHR1 mRNA expression was not different between LA and HA rats in GABAergic neurons (Mann-Whitney test, U = 11.00, P = 0.816) and when quantified in all nuclei (Mann-Whitney test, U = 8.500, P = 0.46). *P < 0.05. Scale bars, 100 µm.

Fig 3: Antisense-mediated CRHR1 down-regulation in the VTA blocks stress effects on PR performance in LA rats.(A) Schematic representation of AON treatment and PR test. (B) Treatment with AONs decreased CRHR1 expression in the VTA (one-tailed t test, t11 = 2.199, P = 0.025). (C) Significant AON treatment × stress interaction, significant treatment effect, and a nonsignificant effect of stress on breakpoint (two-way ANOVA: F1,24 = 6.789, P = 0.016, F1,24 = 10.27, P = 0.004, and F1,24 = 1.872, P = 0.184, respectively, n = 7). (D) Significant AON treatment effect, significant interaction, and nonsignificant effect of stress on the number of correct nosepokes (two-way ANOVA: F1,24 = 9.566, P = 0.005, F1,24 = 6.346, P = 0.019, and F1,24 = 2.146, P = 0.156, respectively, n = 7). (E) Two-way ANOVA revealed a significant AON treatment × stress interaction, a significant effect of AON treatment, and a nonsignificant effect of stress on the number of rewards (F1,24 = 5.526, P = 0.027, F1,24 = 9.566, P = 0.005, and F1,24 = 0.75, P = 0.395, respectively, n = 7). (F) Ex vivo basal firing rate of DA neurons in the VTA, following AON-mediated CRHR1 down-regulation. Treatment with AONs down-regulating CRHR1 resulted in a higher spontaneous firing rate in DA neurons compared to the mismatch treated group (Mann-Whitney test, U = 17.5, P = 0.0222, n = 9 to 10). (G) Bath application of CRH (500 nM) induced a reversible increase in cell firing in LA rats, which was partially blocked by AON treatment (Mann-Whitney test comparing mean values in the shaded area, U = 6, P = 0.0451, n = 5 to 8). Representative voltage traces are shown at the top. Asterisks denote significant differences in the respective post hoc tests (**P < 0.01, *P < 0.05, and #P < 0.1).

Fig 4: Genetic manipulation of CRHR1 expression affects motivated behavior after stress.(A) Schematic representation of the viral strategy for CRHR1 OE or GFP expression in NAc-projecting VTA neurons in HA rats, generating CRHR1 OE rats and control GFP rats, which were tested for PR performance following stress exposure. (B to D) CRHR1 OE rats reached a higher breakpoint (F1,19 = 12.43, P = 0.0027), performed more nosepokes (F1,19 = 13.23, P = 0.0018), and obtained more rewards (F1,19 = 12.06, P = 0.0025) both under basal conditions and after stress. (E) PR performance after stress was tested in DAT-CRHR1 mice in which CRHR1 was selectively down-regulated in DA neurons. (F to H) Stress in DAT-CRHR1 mice led to impaired performance. (F) Stress in DAT-CRHR1 mice led to a lower breakpoint. Two-way repeated-measures ANOVA revealed a significant stress × genotype interaction (F1,20 = 8.028, P = 0.01), a marginally nonsignificant genotype effect, and a nonsignificant stress effect (F1,20 = 4.016, P = 0.059 and F1,20 = 0.004, P = 0.95, n = 7 to 15). (G) Stress reduced the number of correct nosepokes in DAT-CRHR1 mice. Two-way repeated-measures ANOVA revealed a significant stress × genotype interaction (F1,20 = 5.836, P = 0.025), a marginally nonsignificant genotype effect, and a nonsignificant stress effect (F1,20 = 4.013, P = 0.059 and F1,20 = 0.056, P = 0.82, n = 7 to 15). (H) Stress in DAT-CRHR1 mice reduced reward acquisition. Two-way repeated-measures ANOVA revealed a significant stress × genotype interaction (F1,20 = 10.14, P = 0.005), a significant effect of genotype, and a nonsignificant effect of stress (F1,20 = 7.150, P = 0.014 and F1,20 = 0.416, P = 0.53, n = 7 to 15 per group). Asterisks denote significant differences in the t test or post hoc test (**P < 0.01, *P < 0.05, and #P < 0.1).