Mielenz D , Reichel M , Jia T , Quinlan EB , Stöckl T , Mettang M , Zilske D , Kirmizi-Alsan E , Schönberger P , Praetner M , Huber SE , Amato D , Schwarz M , Purohit P , Brachs S , Spranger J , Hess A , Büttner C , Ekici AB , Perez-Branguli F , Winner B , Rauschenberger V , Banaschewski T , Bokde ALW , Büchel C , Conrod PJ , Desrivières S , Flor H , Frouin V , Gallinat J , Garavan H , Gowland P , Heinz A , Martinot JL , Lemaitre H , Nees F , Paus T , Smolka MN , IMAGEN Consortium , Schambony A , Bäuerle T , Eulenburg V , Alzheimer C , Lourdusamy A , Schumann G , Müller CP .

Wellcome Trust , R01 MH085772 NIMH NIH HHS, U54 EB020403 NIBIB NIH HHS , 93558 Medical Research Council , MR/N000390/1 Medical Research Council , Department of Health, G0901858 Medical Research Council

	Figure 2. EFhd2 knockout (KO) mice display a sensation-seeking/low-anxiety behavioural phenotype that is frequently associated with an enhanced risk for alcohol addiction. (a) In the open-field (OF) test EFhd2 KO mice (n=9) show higher locomotor activity in a novel environment than wild-type (WT) mice (n=12). (b) The latency to enter the anxiogenic centre of the maze for the first time is reduced and (c) EFhd2 KO mice spend more time in the centre of the maze than WT mice. (d) Centre locomotion of EFhd2 KO mice is enhanced in the OF. (e) Locomotion in the periphery of the maze is not altered in EFhd2 KO mice. Also the elevated plus maze (EPM) test suggests reduced levels of anxiety in EFhd2 KO (n=8) compared to WT (n=12) mice. (f) The number of entries into the open arms is enhanced in EFhd2 KO mice. (g) Also, locomotion in the open arms is enhanced. In-depth analysis of anxiety-related behaviour in the EPM shows that major differences between EFhd2 KO and WT mice are derived from behaviour in most anxiety-loaded parts of the maze, that is, the distal part of the arms, as shown by (h) the time spent on proximal (Prox) and distal (Dist) arms of the maze, (i) the locomotor activity on all arms of the EPM, and by (j) the entries in the arms of the EPM. (k) In the forced swim test, EFhd2 KO mice show less immobility (floating) than WT mice. (l) The novelty-suppressed feeding (NSF) test shows a strong tendency for reduced depression-like behaviour in EFhd2 KO mice (n=8) compared to WT (n=11) mice. The latency to eat food in a novel environment is largely reduced in EFhd2 KO mice. (m) Speed of locomotion to search out for the food in the NSF test is significantly enhanced in EFhd2 KO mice, indicating a reduced suppression of feeding by the novelty of the environment. (n) The lack of EFhd2 has no effect on sucrose preference (*P<0.05; **P<0.01).
	Figure 3. Low anxiety and high alcohol consumption in EFhd2 knockout (KO) mice can be reversed by chronic subcutaneous (s.c.) treatment with the anxiogenic drug β-carboline-3-carboxylate ethyl ester (β-CCE). β-CCE was administered s.c. by osmotic minipumps at a rate of 1.5 mg kg−1 per day. After 8–9 days of administration, the elevated plus maze (EPM) test revealed a reversal of the low-anxiety phenotype of EFhd2 KO mice (n=7 per group) to the level of wild-type (WT, n=8 and 9 per group) animals. (a) Time spent in the open (OA) and closed arm (CA) or centre (Ctr) of the EPM (*P<0.05). (b) Alcohol consumption in a two-bottle free-choice drinking paradigm in mice with chronic treatment with β-CCE or vehicle (***P<0.001; #P<0.05). (c) Dopamine basal levels in the nucleus accumbens (Nac) and prefrontal cortex (PFC) prior to alcohol treatment. EFhd2 is required for basal dopaminergic tone in the Nac, but not in the PFC of mice (*P<0.05). (d) EFhd2 limits the alcohol-induced increase in extracellular dopamine levels in the Nac, but not in the PFC of mice. Extracellular dopamine levels after acute alcohol (2 g kg−1, intraperitoneal) treatment in EFhd2 KO (n=9) and WT (n=9) mice in (d) the Nac and (e) PFC (*P<0.05, §P<0.01 vs WT).
	Figure 4. EFhd2 controls gene co-expression in the prefrontal cortex (PFC) of mice, a brain region with naturally high EFhd2 expression. (a) Weighted gene co-expression network analysis of mouse PFC expression data comparing alcohol or water drinking EFhd2 knockout (KO) and wild-type (WT) mice (n=4 PER group). Cluster dendrogram generated by hierarchical clustering of genes on the basis of topological overlap. Modules of correlated genes were assigned colours and are indicated by the horizontal bar beneath the dendrogram, where all unassigned genes were placed in the grey module. (b) Expression changes between WT and EFhd2 KO for selected candidate genes (EFhd2, S100a5, Th and Doc2g) of the turquoise model. (c, d) Scatter plot of correlations between gene significance (GS), that is, differential expression between WT and EFhd2 KO (F-statistics from the analysis of variance model) and module membership (MM) for turquoise and green-yellow modules. (e, f) Heatmap of top 100 genes in turquoise and green-yellow modules. In the heatmap, red represents high expression, whereas green represents low expression values.
	Figure 5. EFhd2 controls cortical development and micro-morphology of neuronal cells. (a) In situ hybridization against Efhd2 expression in explanted brains of Xenopus stage 40 tadpoles. A sense probe was used to identify unspecific binding (bar: 0.5 mm). (b) At the eight-cell stage, Xenopus embryos were injected unilaterally into one dorso-animal blastomere with EFhd2 morpholino or an unrelated control morpholino, along with a lacZ-encoding plasmid. The star (*) marks the injected side. At stage 28, β-galactosidase activity was visualized to identify the injected side. Pax6 was detected by whole-mount in situ hybridization (bar: 1 mm). (c) At the eight-cell stage, Xenopus embryos were injected unilaterally into one dorso-animal blastomere with EFhd2 morpholino or an unrelated control morpholino. At stage 28, Eomes expression was assessed by quantitative PCR (***P<0.001). (d) Brain area volume of adult EFhd2 knockout (KO) and wild-type (WT) mice measured with magnetic resonance imaging. Hipp, hippocampus; OB, olfactory bulb; PFC, prefrontal cortex; SMC, sensorimotor cortex; Ventr, ventricle; *P<0.05.
	Figure 6. EFhd2 controls neuronal spine density and dendritic length. (a) Primary cortical neurons were transfected with short hairpin RNAs (shRNAs; scrambled or directed against EFhd2) linked to green fluorescent protein (GFP) expression, with GFP expressing or EFhd2-IRES-GFP vectors, fixed and stained with phalloidine–rhodamine. (b–e) Dendrites and spines of green cells were analysed with ImageJ. Data are shown as mean+s.e.m. (*P<0.05; ***P<0.001).

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