Supplementary Figure 12: Virally mediated expression of a constitutively active (CA) CRHR1-EGFP fusion construct in VTA neurons of Crhr1-ires-Cre mice.

(a) Double ISH against GFP (red staining) and endogenous Crhr1 (silver grains) in Crhr1-ires-Cre mice following VTA-injection of AAV-Ef1α::(CA)CRHR1-GFP (representative coronal sections are shown from rostral to caudal; corresponding atlas images are shown on the left). The vast majority of cells co-express GFP and native Crhr1 (red-framed black arrow), further demonstrating recombination-specificity of the Crhr1-Cre driver. Images are representatives from three independent experiments. (b) Crhr1-ires-Cre mice expressing (CA)CRHR1-EGFP in the VTA exhibit increased locomotion in the OF (unpaired two-tailed t-test: t₍₁₅₎ = 2.45, *p = 0.027), and a trend towards decreased anxiety compared to controls injected with AAV-Ef1α::DIO-mCherry (unpaired two-tailed t-test: inner zone time (%) - t₍₁₅₎ = 1.5, p = 0.15; no. inner zone entries - t₍₁₅₎ = 1.8, p = 0.09; n = 8 Ctrl, 10 (CA)CRHR1). Although locomotion in the open field was increased it is unlikely to compromise the interpretation of the other tests (Fig. 3) given that mice expressing CA(CRHR1) also displayed reduced anxiety in the marble burying task (Fig. 3l), an assay in which confounding increases in locomotion would have produced the opposite effect. (c) No changes between groups were observed during contextual fear conditioning (RM ANOVA – time x genotype interaction: F_(2,34) = 0.45, p = 0.64; genotype: F_(1,17) = 0.05, p = 0.82; n = 8 Ctrl, 11 (CA)CRHR1). Error bars represent s.e.m.