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. 2018 May;23(5):1303-1319.
doi: 10.1038/mp.2017.63. Epub 2017 Apr 11.

EFhd2/Swiprosin-1 Is a Common Genetic Determinator for Sensation-Seeking/Low Anxiety and Alcohol Addiction

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Free PMC article

EFhd2/Swiprosin-1 Is a Common Genetic Determinator for Sensation-Seeking/Low Anxiety and Alcohol Addiction

D Mielenz et al. Mol Psychiatry. .
Free PMC article

Abstract

In many societies, the majority of adults regularly consume alcohol. However, only a small proportion develops alcohol addiction. Individuals at risk often show a high sensation-seeking/low-anxiety behavioural phenotype. Here we asked which role EF hand domain containing 2 (EFhd2; Swiprosin-1) plays in the control of alcohol addiction-associated behaviours. EFhd2 knockout (KO) mice drink more alcohol than controls and spontaneously escalate their consumption. This coincided with a sensation-seeking and low-anxiety phenotype. A reversal of the behavioural phenotype with β-carboline, an anxiogenic inverse benzodiazepine receptor agonist, normalized alcohol preference in EFhd2 KO mice, demonstrating an EFhd2-driven relationship between personality traits and alcohol preference. These findings were confirmed in a human sample where we observed a positive association of the EFhd2 single-nucleotide polymorphism rs112146896 with lifetime drinking and a negative association with anxiety in healthy adolescents. The lack of EFhd2 reduced extracellular dopamine levels in the brain, but enhanced responses to alcohol. In confirmation, gene expression analysis revealed reduced tyrosine hydroxylase expression and the regulation of genes involved in cortex development, Eomes and Pax6, in EFhd2 KO cortices. These findings were corroborated in Xenopus tadpoles by EFhd2 knockdown. Magnetic resonance imaging (MRI) in mice showed that a lack of EFhd2 reduces cortical volume in adults. Moreover, human MRI confirmed the negative association between lifetime alcohol drinking and superior frontal gyrus volume. We propose that EFhd2 is a conserved resilience factor against alcohol consumption and its escalation, working through Pax6/Eomes. Reduced EFhd2 function induces high-risk personality traits of sensation-seeking/low anxiety associated with enhanced alcohol consumption, which may be related to cortex function.

Conflict of interest statement

TB has served as an advisor or consultant to Bristol-Myers Squibb, Desitin Arzneimittel, Eli Lilly, Medice, Novartis, Pfizer, Shire, UCB and Vifor Pharma; he has received conference attendance support, conference support or speaking fees from Eli Lilly, Janssen McNeil, Medice, Novartis, Shire and UCB; and he is involved in clinical trials conducted by Eli Lilly, Novartis and Shire; the present work is unrelated to these relationships. JG has received research funding from the German Federal Ministry of Education and Research, AstraZeneca, Eli Lilly, Janssen-Cilag and Bristol-Myers Squibb; he has received speaking fees from AstraZeneca, Janssen-Cilag and Bristol-Myers Squibb. The remaining authors declare no conflict of interests.

Figures

Figure 1
Figure 1
EFhd2 is a resilience factor for the establishment of alcohol drinking, the escalation of alcohol consumption and the sedating effects of alcohol. EFhd2 knockout (KO; n=11) and wild-type (WT; n=10) mice were tested in a free-choice two-bottle drinking paradigm for their alcohol consumption. (a) Amount of alcohol consumed at different concentrations of the drinking fluid. (b) Preference of alcohol versus water (*P<0.05, **P<0.01; ***P<0.001). (c) Spontaneous escalation of 16 vol. % alcohol consumption after chronic drinking and alcohol deprivation effect (ADE) in EFhd2 KO (n=11) and WT (n=10) mice. After continuous drinking, animals were withdrawn from alcohol for 3 weeks (dotted lines) and reinstated for 4 days. (d) Average alcohol consumption as area under the curve (AUC) 4 days before and after withdrawal indicates an alcohol deprivation effect in WT mice, but not in EFhd2 KO mice ($P<0.05; &P<0.001 vs chronic consumption). (e) Spontaneous escalation in EFhd2 KO mice is confirmed in alcohol preference vs water (*P<0.05, $P<0.01; #P<0.001 vs WT). (f) Sucrose (sweet) preference and quinine (bitter) avoidance test in a free-choice two-bottle drinking paradigm indicates no difference between EFhd2 KO and WT mice in taste preference. EFhd2 KO mice show attenuated sedating effects of alcohol in the loss of righting reflex (LORR) test. (g) Latency to lose the righting reflex and (h) duration of sedation in EFhd2 KO (n=19) and WT (n=23) mice (*P<0.05). (i) Blood alcohol concentration in WT (n=8) and EFhd2 KO mice (n=8) after alcohol injection (3.0 g kg−1, intraperitoneal). Over the 2 h tested, there was no difference in alcohol bioavailability between genotypes (P>0.05).
Figure 2
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
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
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
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
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. (be) 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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