Increased behavioral responses to ethanol in Lmo3 knockout mice

Abstract

LIM-domain-only 3 (LMO3) is a transcriptional regulator involved in central nervous system development and neuroblastoma. Our previous studies implicated a potential role for LMO3 in regulating ethanol sensitivity and consumption. Here, we examined behavioral responses to ethanol in a line of Lmo3 null (Lmo3(Z) ) mice, utilizing the ethanol-induced loss-of-righting-reflex (LORR) test, two-bottle choice ethanol consumption and the drinking in the dark (DID) test, which models binge-like ethanol consumption. We found that Lmo3(Z) mice exhibited increased sedation time in response to ethanol in the LORR test and drank significantly more ethanol in the DID test compared with their wild-type counterparts, but showed no differences in two-bottle choice ethanol consumption. To explore where LMO3 may be acting in the brain to produce an ethanol phenotype, we also examined reporter gene (β-galactosidase) expression in heterozygous Lmo3(Z) mice and found strong expression in subcortical areas, particularly in those areas implicated in drug abuse, including the nucleus accumbens (Acb), cortex, hippocampus and amygdala. We also examined Lmo3 expression in the brains of wild-type mice who had undergone the DID test and found a negative correlation between Lmo3 expression in the Acb and the amount of ethanol consumed, consistent with the increased binge-like drinking observed in Lmo3(Z) mice. These results support a role for LMO3 in regulating behavioral responses to ethanol, potentially through its actions in the Acb.

Keywords: Binge drinking; LMO3; ethanol; sedation.

Figure 1. β-gal reporter expression in the brains of heterozygous Lmo3^Z mice

X-Gal staining of coronal brain sections reveals robust β-gal expression in the (a) caudate putamen (CPu) and nucleus accumbens (Acb), (b) piriform cortex (Pir) and lateral olfactory tract (LOT), (c) basolateral amygdala (BLA) and amygdalostriatal transition area (AStr), (d) paraventricular hypothalamic nucleus (Pa), (e) ventromedial hypothalamic nucleus (VMH), and (f) hippocampus, dentate gyrus (DG), CA1 and CA3 regions. Abbreviations: ac, anterior commissure; 3V, 3^rd ventricle, CeA, central nucleus of the amygdala. Scale bar, 200 microns.

Figure 2. Increased sedation time in response to ethanol in Lmo3^Zmice

Ethanol-induced LORR was tested at 3.2 (a), 3.6 (b), and 4 (c) g/kg ethanol in wild-type (+/+, black bars) and homozygous Lmo3^Z (−/−, white bars) mice. Mice received the indicated doses of ethanol and were tested for the ability to right themselves. Shown is the time to recovery after mice failed to right themselves 3 times within 30 seconds. There was a significant difference between genotypes at 3.2 and 3.6 g/kg ethanol and a significant difference between sexes at 3.6 g/kg ethanol by 2-way ANOVA, *P<0.05. (d) Ethanol clearance from the blood in wild-type (+/+, filled circles) and homozygous Lmo3^Z (−/−, open squares) mice after a single i.p. injection of 4 g/kg ethanol. Shown is the blood ethanol concentration (BEC) over time. No differences were observed in ethanol clearance between genotypes.

Figure 3. Increased binge-like ethanol consumption in Lmo3^Z mice

(a, b) DID during 2 hour drinking sessions on days 1–4 in male (a) and female (b) wild-type (+/+, filled circles) and homozygous (−/−, open squares) Lmo3^Z mice. Lmo3^Z mice drank significantly more ethanol by 2-way RM ANOVA. (c) DID during the 4 hour drinking session on day 4 in wild-type (+/+, black bars) and homozygous Lmo3^Z (−/−, white bars) mice. There were significant effects of genotype, sex, and a genotype by sex interaction by 2-way ANOVA. ^#Significant genotype by sex interaction, P<0.05. (d) Blood ethanol concentrations (BEC, mg%) in wild-type (+/+, black bars) and homozygous Lmo3^Z (−/−, white bars) mice. There was a significant effect of genotype by 2-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Correlation between Lmo3 expression in the Acb and ethanol consumed during DID

Male C57BL/6J mice underwent the DID test with water or ethanol. Mice were euthanized and brain regions dissected 24 hours after the final 4 hour drinking session on day 4. Lmo3 expression was analyzed by qPCR. (a) No significant differences in Lmo3 expression between water and ethanol consumption in the DID test were evident in the CPu, Acb, prefrontal cortex (Pfc), CeA, or BLA. (b) There was a significant correlation between Lmo3 expression in the Acb and the amount of ethanol consumed. (c) There was a trend towards a correlation between Lmo3 expression in the CeA and the amount of ethanol consumed.