Genes, Brain and Behavior (2014) 13: 777–783
doi: 10.1111/gbb.12176
Increased behavioral responses to ethanol in Lmo3 knockout mice A. Savarese† , M. E. Zou‡ , V. Kharazia‡ , R. Maiya‡,§ and A. W. Lasek†,∗ † Department of Psychiatry and Graduate Program in Neuroscience, University of Illinois at Chicago, Chicago, IL, ‡ Department of Neurology, Ernest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA, USA, and § Present address: College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, Austin, TX, USA *Corresponding author: A. W. Lasek, PhD, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor St, M/C 912, Chicago, IL 60612, USA. E-mail: [email protected]
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 (Lmo3Z ) 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 Lmo3Z 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 Lmo3Z 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 Lmo3Z mice. These results support a role for LMO3 in regulating behavioral responses to ethanol, potentially through its actions in the Acb.
interacting with DNA-binding transcription factors through their cysteine-rich zinc-finger LIM domains. LIM-domain-only (LMO) proteins do not bind DNA directly, but instead act as ‘scaffolding’ proteins to form multiprotein complexes that regulate transcription. LMO proteins regulate cell fate determination and differentiation during embryonic development, and all members of this family have been implicated in human cancers (reviewed in Matthews et al. 2013). LMO3 is highly expressed in the mammalian central nervous system during development and in the adult (Bulchand et al. 2003; Hinks et al. 1997; Remedios et al. 2004; Tse et al. 2004). Mice with null mutations of LMO3 are viable, fertile and have no obvious developmental defects (Tse et al. 2004), but have decreased numbers of parvalbumin-positive cortical interneurons, indicating that LMO3 is involved in the specification of basket interneurons in the developing brain (Au et al. 2013). In a search for genes involved in altered behavioral responses to drugs of abuse, we found that Drosophila Lmo (dLmo) regulates acute sensitivity to cocaine (Tsai et al. 2004) and ethanol sedation sensitivity (Lasek et al. 2011b). In addition, expression levels of Lmo3 in mouse brain correlate with two behavioral responses to ethanol, loss-of-righting reflex (LORR) and two-bottle choice consumption (Lasek et al. 2011b). To examine directly whether LMO3 alters behavioral responses to ethanol, we tested Lmo3 null (Lmo3Z ) mice for ethanol-induced LORR, two-bottle choice ethanol consumption and drinking in the dark (DID), a model of binge-drinking behavior. We found that Lmo3 Z mice exhibit increased sensitivity to ethanol sedation and augmented binge-drinking behavior. To begin to understand where LMO3 might function in the brain to regulate behavioral responses to ethanol, we examined reporter gene expression in Lmo3 Z mice because they contain an insertion of the 𝛽-galactosidase (𝛽-gal) gene in the Lmo3 locus driven by the endogenous Lmo3 promoter. Strong 𝛽-gal expression was observed in adult mice in several brain regions that regulate behavioral responses to drugs of abuse, including the nucleus accumbens (Acb), cortex and amygdala. Furthermore, we found that Lmo3 mRNA levels in the Acb of wild-type mice negatively correlated with ethanol consumption in the binge drinking test, suggesting that LMO3 may be acting in the Acb to limit excessive ethanol consumption.
Keywords: Binge drinking, ethanol, LMO3, sedation Received 29 April 2014, revised 15 July 2014 and 20 August 2014, accepted for publication 29 August 2014
LIM-domain-only 3 (LMO3) is a member of a family of four proteins (LMO1–4) that act as transcriptional regulators by
Materials and methods Subjects Lmo3 Z mice containing an IRES-LacZ insertion in exon 2 of Lmo3 have been described previously (Tse et al. 2004). Mice were rederived from targeted embryonic stem cells (E14) and backcrossed two generations into the C57BL/6J background for behavioral testing. Adult (10–16 weeks old) male and female homozygous Lmo3Z
© 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society
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Savarese et al. and wild-type littermates were used for behavioral testing (23 mice for LORR and 31 mice for DID). C57BL/6J mice (29 mice) for gene expression analysis were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were group housed with same-sex cage mates in a temperature- and humidity-controlled environment under a 14-h light/dark cycle (lights on at 0600 h and off at 2000 h) and tested during the light phase, unless they underwent the DID procedure. Mice had access to food and water ad libitum for the duration of the study and were maintained and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures performed on mice were approved by the Ernest Gallo Clinic and Research Center Institutional Animal Care and Use Committee (IACUC) and the University of Illinois at Chicago (UIC) Animal Care Committee (ACC).
𝜷-Gal detection
Heterozygous Lmo3Z mice were evaluated for 𝛽-gal expression in a protocol similar to that used in Tse et al. (2004). Briefly, mice were anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA). Brains were post-fixed for 60 min in 4% PFA and were then placed overnight in 30% sucrose in phosphate-buffered saline. Sections were cut on a sliding microtome to a thickness of 60 μm, placed into a 12-well plate and incubated overnight in X-Gal (5-bromo-4-chloro-3-indolyl-𝛽-D-galactopyranoside; Sigma-Aldrich, St. Louis, MO, USA) at 37∘ C. Sections were mounted on slides and coverslipped with xylene. Images were acquired using a Zeiss AxioScope.A1 microscope fitted with a 5-megapixel AxioCam ERc 5s color camera and analyzed with ZenLite image acquisition and archiving software (Carl Zeiss, Thornwood, NY, USA).
Loss-of-righting reflex Mice were tested for LORR at three doses of ethanol (3.2, 3.6 and 4 g/kg) as described in Lasek et al. (2011c), with 1-week elapsing between doses.
Measurement of blood ethanol concentrations Blood ethanol concentrations (BECs) were measured in the same group of mice that underwent testing for ethanol LORR. One week after the last LORR test, mice were injected intraperitoneally (i.p.) with 4 g/kg ethanol (20% v/v) in 0.9% saline. Blood (20 μl) was collected in heparinized capillary tubes via tail vein puncture at 30, 60, 90, 120 and 180 min after injection. Blood samples were stored at −80∘ C until BECs were determined using a nicotinamide adenine dinucleotide-alcohol dehydrogenase enzymatic assay as described in Zapata et al. (2006). Blood samples were also collected immediately after the last DID session for BEC measurements.
Drinking in the dark Mice were individually housed in a reverse dark cycle room (lights off at 1000 h and on at 2200 h) for at least 2 weeks prior to testing in order to acclimate. Mice were tested for 4-day DID as described in Rhodes et al. (2005) using a 20% ethanol solution in water (v/v). Sipper tubes containing ethanol were placed on the home cage 3 h into the dark cycle for 2 h on Monday, Tuesday and Wednesday and 4 h on Thursday.
Gene expression analysis Brains were rapidly harvested and frozen on dry ice prior to storage at −80∘ C. Sections (200 μm) were cut frozen on a sliding microtome. Tissue was punched out of sections using a 0.74-mm diameter punch from a brain tissue punch set (Thermo Fisher Scientific, Pittsburgh, PA, USA). RNA was isolated using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed using Maxima Probe qPCR Master Mix (Thermo Fisher Scientific), 20× Lmo3 probe/primer mix and 20× mouse Gapdh probe/primer
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mix from Life Technologies (Carlsbad, CA, USA; Lmo3 gene expression assay Mm02392699_m1). Gapdh expression did not change with ethanol treatment.
Statistical analysis All data were analyzed using Prism software version 6.05 (GraphPad, La Jolla, CA, USA). Loss-of-righting-reflex data were analyzed using two-way analysis of variance (ANOVA) (genotype and sex) separately for each dose. Ethanol consumption data (both two-bottle choice and DID) were analyzed using two-way repeated measures (RM) ANOVA for genotype and time. Four-hour DID and BEC data were analyzed using two-way ANOVA for genotype and sex. Post hoc comparisons were performed using the Holm-Sidak test. For gene expression data, individual t-tests were performed to compare Lmo3 expression after water and ethanol exposure within each brain region, and correlations were examined using linear regression.
Results Characterization of 𝜷-gal expression in Lmo3Z mice Lmo3Z mice contain an insertion of the LacZ gene in exon 2 of Lmo3 and therefore express 𝛽-gal from the endogenous Lmo3 promoter. To characterize the putative expression of Lmo3 in mouse brain, adult heterozygous mice were examined for 𝛽-gal reporter expression. 𝛽-Gal was widely detected throughout the brain, predominantly in subcortical areas. Expression was strongest in the Acb, caudate putamen, piriform cortex, specific nuclei of the amygdala, hippocampus and hypothalamus (Fig. 1). Amygdala staining was particularly strong in the lateral olfactory tract and basolateral nucleus (Fig. 1b,c), consistent with Lmo3 expression in the embryonic mouse amygdala (Remedios et al. 2004). In the hippocampus, we observed intense staining in the CA1 and dentate gyrus regions, with lighter staining in the CA3 region (Fig. 1f). Hypothalamic staining was most prominent in the ventromedial hypothalamus and paraventricular nucleus (Fig. 1d,e). 𝛽-Gal was also detected in the septum, habenula, superior colliculus, interpeduncular nucleus, cortex, olfactory tubercle, ventral pallidum and substantia nigra (data not shown). These results are consistent with prior published work examining Lmo3 mRNA expression in the brain using in situ hybridization (Hinks et al. 1997).
LORR in Lmo3Z mice Male and female Lmo3Z mice were tested for ethanol-induced sedation using the LORR test at three doses of ethanol: 3.2, 3.6 and 4 g/kg. At 3.2 (Fig. 2a) and 3.6 g/kg (Fig. 2b) ethanol, Lmo3Z mice spent significantly more time sedated compared with wild-type mice. At 3.2 g/kg ethanol, a two-way ANOVA for genotype and sex indicated a significant main effect of genotype (genotype: F 1,19 = 4.56, P = 0.046; sex: F 1,19 = 0.58, P = 0.46; interaction: F 1,19 = 1.22, P = 0.28). At 3.6 g/kg ethanol, two-way ANOVA demonstrated significant main effects of genotype and sex (genotype: F 1,19 = 6.55, P = 0.019; sex: F 1,19 = 4.45, P = 0.048; interaction: F 1,19 = 0.72, P = 0.41). At 4 g/kg ethanol (Fig. 2c), Lmo3Z mice showed a trend toward increased sedation time (genotype: F 1,20 = 3.30, P = 0.084; sex: F 1,20 = 2.52, P = 0.13; interaction: F 1,20 = 0.022, P = 0.88). Together, these results indicate that male and female Lmo3Z mice are more Genes, Brain and Behavior (2014) 13: 777–783
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Figure 1: 𝛽-Gal reporter expression in the brains of heterozygous Lmo3Z mice. X-Gal staining of coronal brain sections reveals robust 𝛽-gal expression in the (a) caudate putamen (CPu) and 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. ac, anterior commissure; 3V, third ventricle; CeA, central nucleus of the amygdala. Scale bar, 200 μm.
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Ethanol consumption in Lmo3 mice We previously found a positive correlation between levels of Lmo3 in the brain and ethanol intake in the two-bottle choice ethanol consumption test in transgenic mice expressing a short hairpin RNA (shRNA) to Lmo3 (Lasek et al. 2011b). We therefore tested Lmo3Z mice for two-bottle choice ethanol consumption and observed no difference in ethanol consumption or preference in Lmo3Z mice compared with wild-type mice (Fig. S1). Moreover, Lmo3Z mice drank similar amounts of water, saccharin and quinine as wild-type mice in a two-bottle choice test (Fig. S1), indicating that there were no significant differences in overall fluid consumption or taste preference in these mice. We next tested Lmo3Z mice for binge-like ethanol consumption in a 4-day DID procedure. During the 2-h drinking sessions on days 1–4, Lmo3Z mice drank significantly more ethanol than wild-type controls (two-way RM ANOVA, effect of genotype, F 1,29 = 13.90, P = 0.0008; time: F 3,87 = 7.09, P = 0.0003; interaction, F 3,87 = 2.37, P = 0.076). This effect was significant in both males (Fig. 3a, effect of genotype: F 1,15 = 7.98, P = 0.013; time: F 3,45 = 4.17, P = 0.011; interaction: F 3,45 = 0.836, P = 0.481) and females (Fig. 3b, Genes, Brain and Behavior (2014) 13: 777–783
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sensitive to the sedating effect of ethanol. We measured ethanol metabolism in male and female Lmo3Z and wild-type mice to determine whether differences in ethanol clearance might account for the increased sensitivity of these mice to ethanol-induced sedation. Mice were injected i.p. with 4 g/kg ethanol, and blood was collected at 30, 60, 90, 120 and 180 min after injection. No differences were observed in ethanol clearance between male and female mice, so data are collapsed across sex. No genotype differences in ethanol clearance were observed between Lmo3Z and wild-type mice (Fig. 2d), indicating that the LORR phenotype observed in Lmo3Z mice is likely not due to altered ethanol metabolism.
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Figure 2: Increased sedation time in response to ethanol in Lmo3Z mice. 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 Lmo3Z (−/−, 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 three 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 two-way ANOVA, *P < 0.05. (d) Ethanol clearance from the blood in wild-type (+/+, filled circles) and homozygous Lmo3Z (−/−, open squares) mice after a single i.p. injection of 4 g/kg ethanol. Shown is the BEC over time. No differences were observed in ethanol clearance between genotypes.
effect of genotype: F 1,12 = 7.39, P = 0.019; time: F 3,36 = 3.38, P = 0.029; interaction: F 3,36 = 1.47, P = 0.24). Analysis of ethanol consumption during the final 4-h drinking session on day 4 also revealed a significant genotype effect (Lmo3Z
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Figure 3: Increased binge-like ethanol consumption in Lmo3Z mice. DID during 2-h drinking sessions on days 1–4 in male (a) and female (b) wild-type (+/+, filled circles) and homozygous (−/−, open squares) Lmo3Z mice. Lmo3Z mice drank significantly more ethanol by two-way RM ANOVA. (c) DID during the 4-h drinking session on day 4 in wild-type (+/+, black bars) and homozygous Lmo3Z (−/−, white bars) mice. There were significant effects of genotype, sex and a genotype by sex interaction by two-way ANOVA. # Significant genotype by sex interaction, P < 0.05. (d) BEC (mg%) in wild-type (+/+, black bars) and homozygous Lmo3Z (−/−, white bars) mice. There was a significant effect of genotype by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
mice consumed more than wild-type mice), a sex effect (females consumed more than males) and a significant sex by genotype interaction (male and female Lmo3Z and female wild-type mice consumed more than male wild-type mice, Fig. 3c, genotype: F 1,27 = 16.02, P = 0.0004; sex: F 1,27 = 9.88, P = 0.004; genotype by sex interaction, F 1,27 = 5.60, P = 0.025). In agreement with the ethanol consumption data during the final 4-h drinking session, blood ethanol levels were elevated in Lmo3Z mice compared with wild-type mice (Fig. 3d, genotype: F 1,27 = 10.87, P = 0.003; sex: F 1,27 = 7.77, P = 0.01; interaction: F 1,27 = 0.02, P = 0.89). We also tested Lmo3Z and wild-type mice for consumption of a 10% sucrose solution in the DID protocol and found no genotype effect (data not shown), indicating that increased drinking in Lmo3Z mice is specific to ethanol and not a naturally rewarding substance such as sucrose. Together, these data indicate that Lmo3Z mice consume more ethanol in a binge drinking test that results in higher BECs.
Correlation between Lmo3 expression and binge ethanol consumption Because LMO3 appears to regulate ethanol consumption, we tested if ethanol exposure might alter the expression of Lmo3 in specific brain regions or, alternatively, if Lmo3 expression levels correlate with ethanol intake in wild-type C57BL/6J
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mice. We examined Lmo3 gene expression in the Acb, caudate putamen, central nucleus of the amygdala (CeA), basolateral amygdala and the prefrontal cortex 4 h after a single injection of 2 g/kg ethanol. No significant changes in Lmo3 expression were observed under these conditions (R. Maiya and A.W. Lasek, unpublished results). We also examined Lmo3 gene expression in these same brain regions 24 h after the last ethanol or water drinking session in the DID test. Although Lmo3 expression did not change with binge-like ethanol consumption (Fig. 4a), we observed a significant negative correlation (r = −0.51, P = 0.03) between Lmo3 levels in the Acb and ethanol intake 24 h after the last drinking session in the DID test (Fig. 4b). There was also a trend toward a negative correlation (r = −45, P = 0.06) between Lmo3 expression and ethanol consumption in the CeA (Fig. 4c). We did not see a correlation between Lmo3 expression 24 h after the last drinking session and ethanol consumption during the first 3 days of DID (2-h drinking sessions, data not shown), indicating that the correlation was specific to ethanol consumption on the last day of 4-h DID. Together, these data indicate that Lmo3 mRNA expression is not altered by acute or binge-like ethanol exposure, but do suggest that lower levels of Lmo3, due to natural individual variation in expression, correlate with increased binge ethanol consumption. These data are consistent with the increased binge ethanol intake in the DID test observed in Lmo3Z mice.
Discussion This is the first study to demonstrate that behavioral responses to ethanol are increased in Lmo3 null mice. Previously, our laboratory utilized transgenic mice globally expressing a shRNA targeting Lmo3 (shLmo3) and found that decreased expression of Lmo3 correlated with increased ethanol sedation time in the LORR test and reduced two-bottle choice ethanol consumption (Lasek et al. 2011b). Consistent with results obtained with shLmo3 transgenic mice, we found that Lmo3Z mice showed increased sedation time in response to ethanol (Fig. 2), indicating that LMO3 functions to inhibit the sedative effects of ethanol. However, in contrast to the transgenic mice, Lmo3Z mice displayed no difference in two-bottle choice ethanol consumption compared with wild-type mice (Fig. S1). It was surprising to discover that sensitivity to ethanol-induced sedation was comparable between the shLmo3 transgenic and Lmo3Z mice, yet ethanol consumption differed between these mice. It is possible that the complete knockout of Lmo3 expression in Lmo3Z mice, in contrast to modestly reduced Lmo3 expression in the shLmo3 transgenic lines (≤50% reduction), results in compensatory changes during development that normalize moderate ethanol drinking behavior in adulthood. Alternatively, strain effects might account for this discrepancy. shLmo3 transgenic mice were generated in inbred C57BL/6J embryos, while the Lmo3Z mice were backcrossed two generations into the C57BL/6J genetic background and contained approximately 25% of the 129 strain genotype. Genetic polymorphisms between inbred strains of mice can profoundly affect ethanol consumption, Genes, Brain and Behavior (2014) 13: 777–783
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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 h after the final 4-h 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 caudate putamen (CPu), Acb, prefrontal cortex (Pfc), CeA or basolateral amygdala (BLA). (b) There was a significant correlation between Lmo3 expression in the Acb and the amount of ethanol consumed. (c) There was a trend toward a correlation between Lmo3 expression in the CeA and the amount of ethanol consumed.
and 129 mice consume less ethanol than C57BL/6J mice (Bachmanov et al. 1996; Metz et al. 2006). Genetic modifiers in the 129 background could plausibly lessen the impact of Lmo3 knockout on two-bottle choice ethanol consumption in the Lmo3Z mice. The two-bottle choice test is a widely used model of moderate alcohol consumption. However, mice do not reach intoxicating blood ethanol levels (≥0.1%) in this assay. More recently, the DID test was developed to evaluate drinking to intoxication, or binge-like drinking, in which blood ethanol levels greater than 0.1% are achieved (Rhodes et al. 2005). We found that Lmo3Z mice consumed significantly more ethanol than wild-type littermates in the DID test (Fig. 3), indicating Genes, Brain and Behavior (2014) 13: 777–783
that LMO3 functions to curb binge-like ethanol consumption but probably plays a limited role in regulating moderate (i.e. two-bottle choice) ethanol consumption. Notably, a line of mice that have been selectively bred for high drinking in the dark (HDID-1) shows equivalent levels of two-bottle choice drinking at ethanol concentrations of 3–20% compared with the control line (Crabbe et al. 2011), indicating that ethanol consumption in the DID and two-bottle choice tests is genetically separable phenotypes. We observed similar behavioral effects with Alk knockout mice. These mice drink more ethanol in the DID test and remain sedated longer in response to ethanol (Lasek et al. 2011c) but exhibit no difference in two-bottle choice ethanol consumption compared with wild-type mice (A.W. Lasek, unpublished results). The similar phenotypes observed in Lmo3 and Alk knockout mice suggest that these genes may be acting through a common pathway to affect ethanol-related behaviors. Both Lmo3 and Alk are regulated by the homeobox transcription factor ARX (Friocourt & Parnavelas 2011), which is selectively expressed in gamma-Aminobutyric acid (GABA) neurons and functions in cortical GABA interneuron migration (Colasante et al. 2008; Friocourt et al. 2008). Overexpression of LMO3 promotes differentiation of cortical GABA interneurons into the parvalbumin subtype, and Lmo3 null mice exhibit a significant reduction in parvalbumin-positive cortical interneurons, with a concomitant slight increase in somatostatin-positive neurons (Au et al. 2013). Overall, Lmo3 null mice have an equivalent number and normal layer distribution of cortical GABA neurons compared with wild-type mice, yet the ratio of parvalbumin- and somatostatin-expressing neurons is shifted toward the somatostatin subtype in the cortex of Lmo3 null mice. It is not clear how a shift in the ratio of these two subtypes of GABA neurons would affect GABA network signaling. However, modulation of GABA networks by LMO3 could potentially affect acute responses to ethanol and ethanol drinking patterns and thus may underlie the behavioral effects seen in Lmo3Z mice. GABA neurotransmission is known to affect binge-like ethanol consumption (Moore & Boehm 2009; Moore et al. 2007) and ethanol-induced sedation (Liljequist & Engel 1982). In the case of binge-like ethanol consumption, the GABAB receptor agonist baclofen increases DID when administered systemically, whereas the GABAA receptor agonists muscimol and 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride decrease DID (Moore et al. 2007). Because different GABA agonists appear to affect DID differentially, it is difficult to predict whether Lmo3 null mice might have increased or decreased GABA signaling based solely on their DID phenotype. In the case of ethanol-induced sedation, muscimol increases sedation in response to ethanol, while GABA antagonists reduce ethanol sedation in the LORR test (Liljequist & Engel 1982). In addition, blockade of the GABA transporter GAT-1 leads to increased sedation time (Hu et al. 2004). The longer sedation time observed in Lmo3 null mice leads us to hypothesize that these mice might have increased GABA tone. It will be useful to examine GABA signaling and responses to GABA modulators in Lmo3 null mice in future experiments. Where in the brain might LMO3 function to regulate binge-like ethanol consumption? One possibility is the Acb.
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Based on 𝛽-gal reporter expression in Lmo3Z mice, Lmo3 is expressed in the Acb (Fig. 1). We also found that low Lmo3 expression in the Acb correlates with larger amounts of ethanol consumed in the DID test (Fig. 4). The Acb plays a key role in regulating binge-like ethanol consumption (Ben Hamida et al. 2012; Cozzoli et al. 2012; Neasta et al. 2011). Another possibility is that LMO3 acts in the CeA to regulate binge drinking (Cozzoli et al. 2014; Liu et al. 2011; Lowery-Gionta et al. 2012). Although not reaching a statistically significant threshold (P = 0.06), low Lmo3 expression in the CeA also seems to correlate with increased binge-like ethanol intake (Fig. 4). Other brain regions involved in ethanol drinking, such as the prefrontal cortex, caudate putamen and basolateral amygdala, did not show this correlation. It will be interesting to examine GABA neuron subtypes in the Acb and CeA to determine whether the ratios of these cells are altered in Lmo3 null mice similar to what has been observed in the cortex (Au et al. 2013). We found no evidence of ethanol regulating the transcription of Lmo3, at least after acute exposure to a moderate dose of ethanol (2 g/kg) or 1 day after 4 days of binge-like ethanol consumption. We did not examine Lmo3 mRNA levels after a longer exposure to ethanol or at later time points after ethanol withdrawal, so the possibility remains that Lmo3 transcription may change under these conditions. It is also possible that LMO3 protein activity or levels, rather than mRNA expression, may be regulated by ethanol exposure. Future studies are necessary to investigate if this is the case and where LMO3 might function to regulate excessive ethanol consumption. A few LMO3-interacting proteins and transcriptional targets are known, primarily in the context of neuroblastoma development. HEN2, a neuronal basic helix-loop-helix protein, forms a complex with LMO3 to activate transcription of Mash1 (Aoyama et al. 2005; Isogai et al. 2011). LMO3 interacts directly with calcium- and integrin-binding protein (CIB) (Hui et al. 2009) and the tumor suppressor p53 (Larsen et al. 2010). However, the transcription factors that complex with LMO3 to regulate expression of genes in the central nervous system remain unexplored and an important area for future research. LMO3 is not the only LMO family member that alters behavioral responses to drugs of abuse. Prior work in our laboratory demonstrated that mice with reduced levels of LMO4 were more sensitive to the locomotor effects of cocaine, an effect that seems to be driven by its action in the Acb (Lasek et al. 2010). Alk expression is repressed by LMO4 and regulates cocaine sensitization and conditioned place preference in addition to behavioral responses to ethanol (Lasek et al. 2011a). In a genome-wide association study, polymorphisms in Lmo1, which encodes the LMO protein most closely related to LMO3, were found to be associated with the maximum number of alcoholic drinks consumed in a 24-h period (Kapoor et al. 2013). Strikingly, Lmo1 expression is also regulated by the cortical GABA interneuron migration factor ARX (Friocourt & Parnavelas 2011; Lee et al. 2014). These data point to the intriguing possibility that all of the LMO family members regulate behavioral responses to drugs of abuse, potentially through the modulation of GABA neurotransmission.
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Lmo3 regulates ethanol behaviors Lasek, A.W., Kapfhamer, D., Kharazia, V., Gesch, J., Giorgetti, F. & Heberlein, U. (2010) LMO4 in the nucleus accumbens regulates cocaine sensitivity. Genes Brain Behav 9, 817–824. Lasek, A.W., Gesch, J., Giorgetti, F., Kharazia, V. & Heberlein, U. (2011a) Alk is a transcriptional target of LMO4 and ERalpha that promotes cocaine sensitization and reward. J Neurosci 31, 14134–14141. Lasek, A.W., Giorgetti, F., Berger, K.H., Tayor, S. & Heberlein, U. (2011b) Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse. Alcohol Clin Exp Res 35, 1600–1606. Lasek, A.W., Lim, J., Kliethermes, C.L., Berger, K.H., Joslyn, G., Brush, G., Xue, L., Robertson, M., Moore, M.S., Vranizan, K., Morris, S.W., Schuckit, M.A., White, R.L. & Heberlein, U. (2011c) An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol. PLoS One 6, e22636. Lee, K., Mattiske, T., Kitamura, K., Gecz, J. & Shoubridge, C. (2014) Reduced polyalanine-expanded Arx mutant protein in developing mouse subpallium alters Lmo1 transcriptional regulation. Hum Mol Genet 23, 1084–1094. Liljequist, S. & Engel, J. (1982) Effects of GABAergic agonists and antagonists on various ethanol-induced behavioral changes. Psychopharmacology (Berl) 78, 71–75. Liu, J., Yang, A.R., Kelly, T., Puche, A., Esoga, C., June, H.L. Jr., Elnabawi, A., Merchenthaler, I., Sieghart, W., June, H.L. Sr. & Aurelian, L. (2011) Binge alcohol drinking is associated with GABAA alpha2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala. Proc Natl Acad Sci U S A 108, 4465–4470. Lowery-Gionta, E.G., Navarro, M., Li, C., Pleil, K.E., Rinker, J.A., Cox, B.R., Sprow, G.M., Kash, T.L. & Thiele, T.E. (2012) Corticotropin releasing factor signaling in the central amygdala is recruited during binge-like ethanol consumption in C57BL/6J mice. J Neurosci 32, 3405–3413. Matthews, J.M., Lester, K., Joseph, S. & Curtis, D.J. (2013) LIM-domain-only proteins in cancer. Nat Rev Cancer 13, 111–122. Metz, A.V., Chynoweth, J. & Allan, A.M. (2006) Influence of genetic background on alcohol drinking and behavioral phenotypes of 5-HT3 receptor over-expressing mice. Pharmacol Biochem Behav 84, 120–127. Moore, E.M. & Boehm, S.L. 2nd (2009) Site-specific microinjection of baclofen into the anterior ventral tegmental area reduces binge-like ethanol intake in male C57BL/6J mice. Behav Neurosci 123, 555–563. Moore, E.M., Serio, K.M., Goldfarb, K.J., Stepanovska, S., Linsenbardt, D.N. & Boehm, S.L. 2nd (2007) GABAergic modulation of binge-like ethanol intake in C57BL/6J mice. Pharmacol Biochem Behav 88, 105–113. Neasta, J., Ben Hamida, S., Yowell, Q.V., Carnicella, S. & Ron, D. (2011) AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors. Biol Psychiatry 70, 575–582. Remedios, R., Subramanian, L. & Tole, S. (2004) LIM genes parcellate the embryonic amygdala and regulate its development. J Neurosci 24, 6986–6990.
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Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A. & Crabbe, J.C. (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84, 53–63. Tsai, L.T., Bainton, R.J., Blau, J. & Heberlein, U. (2004) Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors. PLoS Biol 2, e408. Tse, E., Smith, A.J., Hunt, S., Lavenir, I., Forster, A., Warren, A.J., Grutz, G., Foroni, L., Carlton, M.B., Colledge, W.H., Boehm, T. & Rabbitts, T.H. (2004) Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice. Mol Cell Biol 24, 2063–2073. Zapata, A., Gonzales, R.A. & Shippenberg, T.S. (2006) Repeated ethanol intoxication induces behavioral sensitization in the absence of a sensitized accumbens dopamine response in C57BL/6J and DBA/2J mice. Neuropsychopharmacology 31, 396–405.
Acknowledgments We thank Terence Rabbitts for Lmo3Z ES cells and Stacy Taylor for assistance in rederiving the mice from ES cells. We also thank Ulrike Heberlein for encouragement and helpful discussions. This work was supported by the National Institute on Alcohol Abuse and Alcoholism, Integrative Neuroscience Initiative on Alcoholism (INIA, U01 AA020912 and AA016654 to A.W.L.) and the Alcohol Center for Translational Genetics at the Ernest Gallo Clinic and Research Center. The authors declare no conflict of interest.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Figure S1: Two-bottle choice ethanol consumption, water consumption and taste preference are not altered in Lmo3Z mice. Male and female Lmo3Z (−/−, white symbols and bars) mice and wild-type (+/+, black symbols and bars) littermates were tested for two-bottle choice ethanol consumption and taste preference after the completion of the LORR test as described in Lasek et al. (2011b). (a) Amount of ethanol consumed (in g/kg/day) at 3%, 6%, 10%, 14% and 20% ethanol (v/v). (b) Ethanol preference expressed as a ratio based on total fluid consumption at the indicated concentrations of ethanol. (c) Amount of water consumed, expressed as g/kg/day. (d) Two-bottle choice taste test for sweet (saccharin, 0.03% and 0.06%) and bitter (quinine, 0.015 mM and 0.03 mM) solutions, expressed as a preference ratio based on total fluid consumption. No significant differences were found in any of the measures tested.
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doi: 10.1111/gbb.12176
Increased behavioral responses to ethanol in Lmo3 knockout mice A. Savarese† , M. E. Zou‡ , V. Kharazia‡ , R. Maiya‡,§ and A. W. Lasek†,∗ † Department of Psychiatry and Graduate Program in Neuroscience, University of Illinois at Chicago, Chicago, IL, ‡ Department of Neurology, Ernest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA, USA, and § Present address: College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, Austin, TX, USA *Corresponding author: A. W. Lasek, PhD, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor St, M/C 912, Chicago, IL 60612, USA. E-mail: [email protected]
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 (Lmo3Z ) 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 Lmo3Z 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 Lmo3Z 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 Lmo3Z mice. These results support a role for LMO3 in regulating behavioral responses to ethanol, potentially through its actions in the Acb.
interacting with DNA-binding transcription factors through their cysteine-rich zinc-finger LIM domains. LIM-domain-only (LMO) proteins do not bind DNA directly, but instead act as ‘scaffolding’ proteins to form multiprotein complexes that regulate transcription. LMO proteins regulate cell fate determination and differentiation during embryonic development, and all members of this family have been implicated in human cancers (reviewed in Matthews et al. 2013). LMO3 is highly expressed in the mammalian central nervous system during development and in the adult (Bulchand et al. 2003; Hinks et al. 1997; Remedios et al. 2004; Tse et al. 2004). Mice with null mutations of LMO3 are viable, fertile and have no obvious developmental defects (Tse et al. 2004), but have decreased numbers of parvalbumin-positive cortical interneurons, indicating that LMO3 is involved in the specification of basket interneurons in the developing brain (Au et al. 2013). In a search for genes involved in altered behavioral responses to drugs of abuse, we found that Drosophila Lmo (dLmo) regulates acute sensitivity to cocaine (Tsai et al. 2004) and ethanol sedation sensitivity (Lasek et al. 2011b). In addition, expression levels of Lmo3 in mouse brain correlate with two behavioral responses to ethanol, loss-of-righting reflex (LORR) and two-bottle choice consumption (Lasek et al. 2011b). To examine directly whether LMO3 alters behavioral responses to ethanol, we tested Lmo3 null (Lmo3Z ) mice for ethanol-induced LORR, two-bottle choice ethanol consumption and drinking in the dark (DID), a model of binge-drinking behavior. We found that Lmo3 Z mice exhibit increased sensitivity to ethanol sedation and augmented binge-drinking behavior. To begin to understand where LMO3 might function in the brain to regulate behavioral responses to ethanol, we examined reporter gene expression in Lmo3 Z mice because they contain an insertion of the 𝛽-galactosidase (𝛽-gal) gene in the Lmo3 locus driven by the endogenous Lmo3 promoter. Strong 𝛽-gal expression was observed in adult mice in several brain regions that regulate behavioral responses to drugs of abuse, including the nucleus accumbens (Acb), cortex and amygdala. Furthermore, we found that Lmo3 mRNA levels in the Acb of wild-type mice negatively correlated with ethanol consumption in the binge drinking test, suggesting that LMO3 may be acting in the Acb to limit excessive ethanol consumption.
Keywords: Binge drinking, ethanol, LMO3, sedation Received 29 April 2014, revised 15 July 2014 and 20 August 2014, accepted for publication 29 August 2014
LIM-domain-only 3 (LMO3) is a member of a family of four proteins (LMO1–4) that act as transcriptional regulators by
Materials and methods Subjects Lmo3 Z mice containing an IRES-LacZ insertion in exon 2 of Lmo3 have been described previously (Tse et al. 2004). Mice were rederived from targeted embryonic stem cells (E14) and backcrossed two generations into the C57BL/6J background for behavioral testing. Adult (10–16 weeks old) male and female homozygous Lmo3Z
© 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society
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Savarese et al. and wild-type littermates were used for behavioral testing (23 mice for LORR and 31 mice for DID). C57BL/6J mice (29 mice) for gene expression analysis were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were group housed with same-sex cage mates in a temperature- and humidity-controlled environment under a 14-h light/dark cycle (lights on at 0600 h and off at 2000 h) and tested during the light phase, unless they underwent the DID procedure. Mice had access to food and water ad libitum for the duration of the study and were maintained and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures performed on mice were approved by the Ernest Gallo Clinic and Research Center Institutional Animal Care and Use Committee (IACUC) and the University of Illinois at Chicago (UIC) Animal Care Committee (ACC).
𝜷-Gal detection
Heterozygous Lmo3Z mice were evaluated for 𝛽-gal expression in a protocol similar to that used in Tse et al. (2004). Briefly, mice were anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA). Brains were post-fixed for 60 min in 4% PFA and were then placed overnight in 30% sucrose in phosphate-buffered saline. Sections were cut on a sliding microtome to a thickness of 60 μm, placed into a 12-well plate and incubated overnight in X-Gal (5-bromo-4-chloro-3-indolyl-𝛽-D-galactopyranoside; Sigma-Aldrich, St. Louis, MO, USA) at 37∘ C. Sections were mounted on slides and coverslipped with xylene. Images were acquired using a Zeiss AxioScope.A1 microscope fitted with a 5-megapixel AxioCam ERc 5s color camera and analyzed with ZenLite image acquisition and archiving software (Carl Zeiss, Thornwood, NY, USA).
Loss-of-righting reflex Mice were tested for LORR at three doses of ethanol (3.2, 3.6 and 4 g/kg) as described in Lasek et al. (2011c), with 1-week elapsing between doses.
Measurement of blood ethanol concentrations Blood ethanol concentrations (BECs) were measured in the same group of mice that underwent testing for ethanol LORR. One week after the last LORR test, mice were injected intraperitoneally (i.p.) with 4 g/kg ethanol (20% v/v) in 0.9% saline. Blood (20 μl) was collected in heparinized capillary tubes via tail vein puncture at 30, 60, 90, 120 and 180 min after injection. Blood samples were stored at −80∘ C until BECs were determined using a nicotinamide adenine dinucleotide-alcohol dehydrogenase enzymatic assay as described in Zapata et al. (2006). Blood samples were also collected immediately after the last DID session for BEC measurements.
Drinking in the dark Mice were individually housed in a reverse dark cycle room (lights off at 1000 h and on at 2200 h) for at least 2 weeks prior to testing in order to acclimate. Mice were tested for 4-day DID as described in Rhodes et al. (2005) using a 20% ethanol solution in water (v/v). Sipper tubes containing ethanol were placed on the home cage 3 h into the dark cycle for 2 h on Monday, Tuesday and Wednesday and 4 h on Thursday.
Gene expression analysis Brains were rapidly harvested and frozen on dry ice prior to storage at −80∘ C. Sections (200 μm) were cut frozen on a sliding microtome. Tissue was punched out of sections using a 0.74-mm diameter punch from a brain tissue punch set (Thermo Fisher Scientific, Pittsburgh, PA, USA). RNA was isolated using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed using Maxima Probe qPCR Master Mix (Thermo Fisher Scientific), 20× Lmo3 probe/primer mix and 20× mouse Gapdh probe/primer
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mix from Life Technologies (Carlsbad, CA, USA; Lmo3 gene expression assay Mm02392699_m1). Gapdh expression did not change with ethanol treatment.
Statistical analysis All data were analyzed using Prism software version 6.05 (GraphPad, La Jolla, CA, USA). Loss-of-righting-reflex data were analyzed using two-way analysis of variance (ANOVA) (genotype and sex) separately for each dose. Ethanol consumption data (both two-bottle choice and DID) were analyzed using two-way repeated measures (RM) ANOVA for genotype and time. Four-hour DID and BEC data were analyzed using two-way ANOVA for genotype and sex. Post hoc comparisons were performed using the Holm-Sidak test. For gene expression data, individual t-tests were performed to compare Lmo3 expression after water and ethanol exposure within each brain region, and correlations were examined using linear regression.
Results Characterization of 𝜷-gal expression in Lmo3Z mice Lmo3Z mice contain an insertion of the LacZ gene in exon 2 of Lmo3 and therefore express 𝛽-gal from the endogenous Lmo3 promoter. To characterize the putative expression of Lmo3 in mouse brain, adult heterozygous mice were examined for 𝛽-gal reporter expression. 𝛽-Gal was widely detected throughout the brain, predominantly in subcortical areas. Expression was strongest in the Acb, caudate putamen, piriform cortex, specific nuclei of the amygdala, hippocampus and hypothalamus (Fig. 1). Amygdala staining was particularly strong in the lateral olfactory tract and basolateral nucleus (Fig. 1b,c), consistent with Lmo3 expression in the embryonic mouse amygdala (Remedios et al. 2004). In the hippocampus, we observed intense staining in the CA1 and dentate gyrus regions, with lighter staining in the CA3 region (Fig. 1f). Hypothalamic staining was most prominent in the ventromedial hypothalamus and paraventricular nucleus (Fig. 1d,e). 𝛽-Gal was also detected in the septum, habenula, superior colliculus, interpeduncular nucleus, cortex, olfactory tubercle, ventral pallidum and substantia nigra (data not shown). These results are consistent with prior published work examining Lmo3 mRNA expression in the brain using in situ hybridization (Hinks et al. 1997).
LORR in Lmo3Z mice Male and female Lmo3Z mice were tested for ethanol-induced sedation using the LORR test at three doses of ethanol: 3.2, 3.6 and 4 g/kg. At 3.2 (Fig. 2a) and 3.6 g/kg (Fig. 2b) ethanol, Lmo3Z mice spent significantly more time sedated compared with wild-type mice. At 3.2 g/kg ethanol, a two-way ANOVA for genotype and sex indicated a significant main effect of genotype (genotype: F 1,19 = 4.56, P = 0.046; sex: F 1,19 = 0.58, P = 0.46; interaction: F 1,19 = 1.22, P = 0.28). At 3.6 g/kg ethanol, two-way ANOVA demonstrated significant main effects of genotype and sex (genotype: F 1,19 = 6.55, P = 0.019; sex: F 1,19 = 4.45, P = 0.048; interaction: F 1,19 = 0.72, P = 0.41). At 4 g/kg ethanol (Fig. 2c), Lmo3Z mice showed a trend toward increased sedation time (genotype: F 1,20 = 3.30, P = 0.084; sex: F 1,20 = 2.52, P = 0.13; interaction: F 1,20 = 0.022, P = 0.88). Together, these results indicate that male and female Lmo3Z mice are more Genes, Brain and Behavior (2014) 13: 777–783
Lmo3 regulates ethanol behaviors
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Figure 1: 𝛽-Gal reporter expression in the brains of heterozygous Lmo3Z mice. X-Gal staining of coronal brain sections reveals robust 𝛽-gal expression in the (a) caudate putamen (CPu) and 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. ac, anterior commissure; 3V, third ventricle; CeA, central nucleus of the amygdala. Scale bar, 200 μm.
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Ethanol consumption in Lmo3 mice We previously found a positive correlation between levels of Lmo3 in the brain and ethanol intake in the two-bottle choice ethanol consumption test in transgenic mice expressing a short hairpin RNA (shRNA) to Lmo3 (Lasek et al. 2011b). We therefore tested Lmo3Z mice for two-bottle choice ethanol consumption and observed no difference in ethanol consumption or preference in Lmo3Z mice compared with wild-type mice (Fig. S1). Moreover, Lmo3Z mice drank similar amounts of water, saccharin and quinine as wild-type mice in a two-bottle choice test (Fig. S1), indicating that there were no significant differences in overall fluid consumption or taste preference in these mice. We next tested Lmo3Z mice for binge-like ethanol consumption in a 4-day DID procedure. During the 2-h drinking sessions on days 1–4, Lmo3Z mice drank significantly more ethanol than wild-type controls (two-way RM ANOVA, effect of genotype, F 1,29 = 13.90, P = 0.0008; time: F 3,87 = 7.09, P = 0.0003; interaction, F 3,87 = 2.37, P = 0.076). This effect was significant in both males (Fig. 3a, effect of genotype: F 1,15 = 7.98, P = 0.013; time: F 3,45 = 4.17, P = 0.011; interaction: F 3,45 = 0.836, P = 0.481) and females (Fig. 3b, Genes, Brain and Behavior (2014) 13: 777–783
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sensitive to the sedating effect of ethanol. We measured ethanol metabolism in male and female Lmo3Z and wild-type mice to determine whether differences in ethanol clearance might account for the increased sensitivity of these mice to ethanol-induced sedation. Mice were injected i.p. with 4 g/kg ethanol, and blood was collected at 30, 60, 90, 120 and 180 min after injection. No differences were observed in ethanol clearance between male and female mice, so data are collapsed across sex. No genotype differences in ethanol clearance were observed between Lmo3Z and wild-type mice (Fig. 2d), indicating that the LORR phenotype observed in Lmo3Z mice is likely not due to altered ethanol metabolism.
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Figure 2: Increased sedation time in response to ethanol in Lmo3Z mice. 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 Lmo3Z (−/−, 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 three 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 two-way ANOVA, *P < 0.05. (d) Ethanol clearance from the blood in wild-type (+/+, filled circles) and homozygous Lmo3Z (−/−, open squares) mice after a single i.p. injection of 4 g/kg ethanol. Shown is the BEC over time. No differences were observed in ethanol clearance between genotypes.
effect of genotype: F 1,12 = 7.39, P = 0.019; time: F 3,36 = 3.38, P = 0.029; interaction: F 3,36 = 1.47, P = 0.24). Analysis of ethanol consumption during the final 4-h drinking session on day 4 also revealed a significant genotype effect (Lmo3Z
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Figure 3: Increased binge-like ethanol consumption in Lmo3Z mice. DID during 2-h drinking sessions on days 1–4 in male (a) and female (b) wild-type (+/+, filled circles) and homozygous (−/−, open squares) Lmo3Z mice. Lmo3Z mice drank significantly more ethanol by two-way RM ANOVA. (c) DID during the 4-h drinking session on day 4 in wild-type (+/+, black bars) and homozygous Lmo3Z (−/−, white bars) mice. There were significant effects of genotype, sex and a genotype by sex interaction by two-way ANOVA. # Significant genotype by sex interaction, P < 0.05. (d) BEC (mg%) in wild-type (+/+, black bars) and homozygous Lmo3Z (−/−, white bars) mice. There was a significant effect of genotype by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
mice consumed more than wild-type mice), a sex effect (females consumed more than males) and a significant sex by genotype interaction (male and female Lmo3Z and female wild-type mice consumed more than male wild-type mice, Fig. 3c, genotype: F 1,27 = 16.02, P = 0.0004; sex: F 1,27 = 9.88, P = 0.004; genotype by sex interaction, F 1,27 = 5.60, P = 0.025). In agreement with the ethanol consumption data during the final 4-h drinking session, blood ethanol levels were elevated in Lmo3Z mice compared with wild-type mice (Fig. 3d, genotype: F 1,27 = 10.87, P = 0.003; sex: F 1,27 = 7.77, P = 0.01; interaction: F 1,27 = 0.02, P = 0.89). We also tested Lmo3Z and wild-type mice for consumption of a 10% sucrose solution in the DID protocol and found no genotype effect (data not shown), indicating that increased drinking in Lmo3Z mice is specific to ethanol and not a naturally rewarding substance such as sucrose. Together, these data indicate that Lmo3Z mice consume more ethanol in a binge drinking test that results in higher BECs.
Correlation between Lmo3 expression and binge ethanol consumption Because LMO3 appears to regulate ethanol consumption, we tested if ethanol exposure might alter the expression of Lmo3 in specific brain regions or, alternatively, if Lmo3 expression levels correlate with ethanol intake in wild-type C57BL/6J
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mice. We examined Lmo3 gene expression in the Acb, caudate putamen, central nucleus of the amygdala (CeA), basolateral amygdala and the prefrontal cortex 4 h after a single injection of 2 g/kg ethanol. No significant changes in Lmo3 expression were observed under these conditions (R. Maiya and A.W. Lasek, unpublished results). We also examined Lmo3 gene expression in these same brain regions 24 h after the last ethanol or water drinking session in the DID test. Although Lmo3 expression did not change with binge-like ethanol consumption (Fig. 4a), we observed a significant negative correlation (r = −0.51, P = 0.03) between Lmo3 levels in the Acb and ethanol intake 24 h after the last drinking session in the DID test (Fig. 4b). There was also a trend toward a negative correlation (r = −45, P = 0.06) between Lmo3 expression and ethanol consumption in the CeA (Fig. 4c). We did not see a correlation between Lmo3 expression 24 h after the last drinking session and ethanol consumption during the first 3 days of DID (2-h drinking sessions, data not shown), indicating that the correlation was specific to ethanol consumption on the last day of 4-h DID. Together, these data indicate that Lmo3 mRNA expression is not altered by acute or binge-like ethanol exposure, but do suggest that lower levels of Lmo3, due to natural individual variation in expression, correlate with increased binge ethanol consumption. These data are consistent with the increased binge ethanol intake in the DID test observed in Lmo3Z mice.
Discussion This is the first study to demonstrate that behavioral responses to ethanol are increased in Lmo3 null mice. Previously, our laboratory utilized transgenic mice globally expressing a shRNA targeting Lmo3 (shLmo3) and found that decreased expression of Lmo3 correlated with increased ethanol sedation time in the LORR test and reduced two-bottle choice ethanol consumption (Lasek et al. 2011b). Consistent with results obtained with shLmo3 transgenic mice, we found that Lmo3Z mice showed increased sedation time in response to ethanol (Fig. 2), indicating that LMO3 functions to inhibit the sedative effects of ethanol. However, in contrast to the transgenic mice, Lmo3Z mice displayed no difference in two-bottle choice ethanol consumption compared with wild-type mice (Fig. S1). It was surprising to discover that sensitivity to ethanol-induced sedation was comparable between the shLmo3 transgenic and Lmo3Z mice, yet ethanol consumption differed between these mice. It is possible that the complete knockout of Lmo3 expression in Lmo3Z mice, in contrast to modestly reduced Lmo3 expression in the shLmo3 transgenic lines (≤50% reduction), results in compensatory changes during development that normalize moderate ethanol drinking behavior in adulthood. Alternatively, strain effects might account for this discrepancy. shLmo3 transgenic mice were generated in inbred C57BL/6J embryos, while the Lmo3Z mice were backcrossed two generations into the C57BL/6J genetic background and contained approximately 25% of the 129 strain genotype. Genetic polymorphisms between inbred strains of mice can profoundly affect ethanol consumption, Genes, Brain and Behavior (2014) 13: 777–783
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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 h after the final 4-h 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 caudate putamen (CPu), Acb, prefrontal cortex (Pfc), CeA or basolateral amygdala (BLA). (b) There was a significant correlation between Lmo3 expression in the Acb and the amount of ethanol consumed. (c) There was a trend toward a correlation between Lmo3 expression in the CeA and the amount of ethanol consumed.
and 129 mice consume less ethanol than C57BL/6J mice (Bachmanov et al. 1996; Metz et al. 2006). Genetic modifiers in the 129 background could plausibly lessen the impact of Lmo3 knockout on two-bottle choice ethanol consumption in the Lmo3Z mice. The two-bottle choice test is a widely used model of moderate alcohol consumption. However, mice do not reach intoxicating blood ethanol levels (≥0.1%) in this assay. More recently, the DID test was developed to evaluate drinking to intoxication, or binge-like drinking, in which blood ethanol levels greater than 0.1% are achieved (Rhodes et al. 2005). We found that Lmo3Z mice consumed significantly more ethanol than wild-type littermates in the DID test (Fig. 3), indicating Genes, Brain and Behavior (2014) 13: 777–783
that LMO3 functions to curb binge-like ethanol consumption but probably plays a limited role in regulating moderate (i.e. two-bottle choice) ethanol consumption. Notably, a line of mice that have been selectively bred for high drinking in the dark (HDID-1) shows equivalent levels of two-bottle choice drinking at ethanol concentrations of 3–20% compared with the control line (Crabbe et al. 2011), indicating that ethanol consumption in the DID and two-bottle choice tests is genetically separable phenotypes. We observed similar behavioral effects with Alk knockout mice. These mice drink more ethanol in the DID test and remain sedated longer in response to ethanol (Lasek et al. 2011c) but exhibit no difference in two-bottle choice ethanol consumption compared with wild-type mice (A.W. Lasek, unpublished results). The similar phenotypes observed in Lmo3 and Alk knockout mice suggest that these genes may be acting through a common pathway to affect ethanol-related behaviors. Both Lmo3 and Alk are regulated by the homeobox transcription factor ARX (Friocourt & Parnavelas 2011), which is selectively expressed in gamma-Aminobutyric acid (GABA) neurons and functions in cortical GABA interneuron migration (Colasante et al. 2008; Friocourt et al. 2008). Overexpression of LMO3 promotes differentiation of cortical GABA interneurons into the parvalbumin subtype, and Lmo3 null mice exhibit a significant reduction in parvalbumin-positive cortical interneurons, with a concomitant slight increase in somatostatin-positive neurons (Au et al. 2013). Overall, Lmo3 null mice have an equivalent number and normal layer distribution of cortical GABA neurons compared with wild-type mice, yet the ratio of parvalbumin- and somatostatin-expressing neurons is shifted toward the somatostatin subtype in the cortex of Lmo3 null mice. It is not clear how a shift in the ratio of these two subtypes of GABA neurons would affect GABA network signaling. However, modulation of GABA networks by LMO3 could potentially affect acute responses to ethanol and ethanol drinking patterns and thus may underlie the behavioral effects seen in Lmo3Z mice. GABA neurotransmission is known to affect binge-like ethanol consumption (Moore & Boehm 2009; Moore et al. 2007) and ethanol-induced sedation (Liljequist & Engel 1982). In the case of binge-like ethanol consumption, the GABAB receptor agonist baclofen increases DID when administered systemically, whereas the GABAA receptor agonists muscimol and 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride decrease DID (Moore et al. 2007). Because different GABA agonists appear to affect DID differentially, it is difficult to predict whether Lmo3 null mice might have increased or decreased GABA signaling based solely on their DID phenotype. In the case of ethanol-induced sedation, muscimol increases sedation in response to ethanol, while GABA antagonists reduce ethanol sedation in the LORR test (Liljequist & Engel 1982). In addition, blockade of the GABA transporter GAT-1 leads to increased sedation time (Hu et al. 2004). The longer sedation time observed in Lmo3 null mice leads us to hypothesize that these mice might have increased GABA tone. It will be useful to examine GABA signaling and responses to GABA modulators in Lmo3 null mice in future experiments. Where in the brain might LMO3 function to regulate binge-like ethanol consumption? One possibility is the Acb.
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Based on 𝛽-gal reporter expression in Lmo3Z mice, Lmo3 is expressed in the Acb (Fig. 1). We also found that low Lmo3 expression in the Acb correlates with larger amounts of ethanol consumed in the DID test (Fig. 4). The Acb plays a key role in regulating binge-like ethanol consumption (Ben Hamida et al. 2012; Cozzoli et al. 2012; Neasta et al. 2011). Another possibility is that LMO3 acts in the CeA to regulate binge drinking (Cozzoli et al. 2014; Liu et al. 2011; Lowery-Gionta et al. 2012). Although not reaching a statistically significant threshold (P = 0.06), low Lmo3 expression in the CeA also seems to correlate with increased binge-like ethanol intake (Fig. 4). Other brain regions involved in ethanol drinking, such as the prefrontal cortex, caudate putamen and basolateral amygdala, did not show this correlation. It will be interesting to examine GABA neuron subtypes in the Acb and CeA to determine whether the ratios of these cells are altered in Lmo3 null mice similar to what has been observed in the cortex (Au et al. 2013). We found no evidence of ethanol regulating the transcription of Lmo3, at least after acute exposure to a moderate dose of ethanol (2 g/kg) or 1 day after 4 days of binge-like ethanol consumption. We did not examine Lmo3 mRNA levels after a longer exposure to ethanol or at later time points after ethanol withdrawal, so the possibility remains that Lmo3 transcription may change under these conditions. It is also possible that LMO3 protein activity or levels, rather than mRNA expression, may be regulated by ethanol exposure. Future studies are necessary to investigate if this is the case and where LMO3 might function to regulate excessive ethanol consumption. A few LMO3-interacting proteins and transcriptional targets are known, primarily in the context of neuroblastoma development. HEN2, a neuronal basic helix-loop-helix protein, forms a complex with LMO3 to activate transcription of Mash1 (Aoyama et al. 2005; Isogai et al. 2011). LMO3 interacts directly with calcium- and integrin-binding protein (CIB) (Hui et al. 2009) and the tumor suppressor p53 (Larsen et al. 2010). However, the transcription factors that complex with LMO3 to regulate expression of genes in the central nervous system remain unexplored and an important area for future research. LMO3 is not the only LMO family member that alters behavioral responses to drugs of abuse. Prior work in our laboratory demonstrated that mice with reduced levels of LMO4 were more sensitive to the locomotor effects of cocaine, an effect that seems to be driven by its action in the Acb (Lasek et al. 2010). Alk expression is repressed by LMO4 and regulates cocaine sensitization and conditioned place preference in addition to behavioral responses to ethanol (Lasek et al. 2011a). In a genome-wide association study, polymorphisms in Lmo1, which encodes the LMO protein most closely related to LMO3, were found to be associated with the maximum number of alcoholic drinks consumed in a 24-h period (Kapoor et al. 2013). Strikingly, Lmo1 expression is also regulated by the cortical GABA interneuron migration factor ARX (Friocourt & Parnavelas 2011; Lee et al. 2014). These data point to the intriguing possibility that all of the LMO family members regulate behavioral responses to drugs of abuse, potentially through the modulation of GABA neurotransmission.
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Lmo3 regulates ethanol behaviors Lasek, A.W., Kapfhamer, D., Kharazia, V., Gesch, J., Giorgetti, F. & Heberlein, U. (2010) LMO4 in the nucleus accumbens regulates cocaine sensitivity. Genes Brain Behav 9, 817–824. Lasek, A.W., Gesch, J., Giorgetti, F., Kharazia, V. & Heberlein, U. (2011a) Alk is a transcriptional target of LMO4 and ERalpha that promotes cocaine sensitization and reward. J Neurosci 31, 14134–14141. Lasek, A.W., Giorgetti, F., Berger, K.H., Tayor, S. & Heberlein, U. (2011b) Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse. Alcohol Clin Exp Res 35, 1600–1606. Lasek, A.W., Lim, J., Kliethermes, C.L., Berger, K.H., Joslyn, G., Brush, G., Xue, L., Robertson, M., Moore, M.S., Vranizan, K., Morris, S.W., Schuckit, M.A., White, R.L. & Heberlein, U. (2011c) An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol. PLoS One 6, e22636. Lee, K., Mattiske, T., Kitamura, K., Gecz, J. & Shoubridge, C. (2014) Reduced polyalanine-expanded Arx mutant protein in developing mouse subpallium alters Lmo1 transcriptional regulation. Hum Mol Genet 23, 1084–1094. Liljequist, S. & Engel, J. (1982) Effects of GABAergic agonists and antagonists on various ethanol-induced behavioral changes. Psychopharmacology (Berl) 78, 71–75. Liu, J., Yang, A.R., Kelly, T., Puche, A., Esoga, C., June, H.L. Jr., Elnabawi, A., Merchenthaler, I., Sieghart, W., June, H.L. Sr. & Aurelian, L. (2011) Binge alcohol drinking is associated with GABAA alpha2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala. Proc Natl Acad Sci U S A 108, 4465–4470. Lowery-Gionta, E.G., Navarro, M., Li, C., Pleil, K.E., Rinker, J.A., Cox, B.R., Sprow, G.M., Kash, T.L. & Thiele, T.E. (2012) Corticotropin releasing factor signaling in the central amygdala is recruited during binge-like ethanol consumption in C57BL/6J mice. J Neurosci 32, 3405–3413. Matthews, J.M., Lester, K., Joseph, S. & Curtis, D.J. (2013) LIM-domain-only proteins in cancer. Nat Rev Cancer 13, 111–122. Metz, A.V., Chynoweth, J. & Allan, A.M. (2006) Influence of genetic background on alcohol drinking and behavioral phenotypes of 5-HT3 receptor over-expressing mice. Pharmacol Biochem Behav 84, 120–127. Moore, E.M. & Boehm, S.L. 2nd (2009) Site-specific microinjection of baclofen into the anterior ventral tegmental area reduces binge-like ethanol intake in male C57BL/6J mice. Behav Neurosci 123, 555–563. Moore, E.M., Serio, K.M., Goldfarb, K.J., Stepanovska, S., Linsenbardt, D.N. & Boehm, S.L. 2nd (2007) GABAergic modulation of binge-like ethanol intake in C57BL/6J mice. Pharmacol Biochem Behav 88, 105–113. Neasta, J., Ben Hamida, S., Yowell, Q.V., Carnicella, S. & Ron, D. (2011) AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors. Biol Psychiatry 70, 575–582. Remedios, R., Subramanian, L. & Tole, S. (2004) LIM genes parcellate the embryonic amygdala and regulate its development. J Neurosci 24, 6986–6990.
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Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A. & Crabbe, J.C. (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84, 53–63. Tsai, L.T., Bainton, R.J., Blau, J. & Heberlein, U. (2004) Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors. PLoS Biol 2, e408. Tse, E., Smith, A.J., Hunt, S., Lavenir, I., Forster, A., Warren, A.J., Grutz, G., Foroni, L., Carlton, M.B., Colledge, W.H., Boehm, T. & Rabbitts, T.H. (2004) Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice. Mol Cell Biol 24, 2063–2073. Zapata, A., Gonzales, R.A. & Shippenberg, T.S. (2006) Repeated ethanol intoxication induces behavioral sensitization in the absence of a sensitized accumbens dopamine response in C57BL/6J and DBA/2J mice. Neuropsychopharmacology 31, 396–405.
Acknowledgments We thank Terence Rabbitts for Lmo3Z ES cells and Stacy Taylor for assistance in rederiving the mice from ES cells. We also thank Ulrike Heberlein for encouragement and helpful discussions. This work was supported by the National Institute on Alcohol Abuse and Alcoholism, Integrative Neuroscience Initiative on Alcoholism (INIA, U01 AA020912 and AA016654 to A.W.L.) and the Alcohol Center for Translational Genetics at the Ernest Gallo Clinic and Research Center. The authors declare no conflict of interest.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Figure S1: Two-bottle choice ethanol consumption, water consumption and taste preference are not altered in Lmo3Z mice. Male and female Lmo3Z (−/−, white symbols and bars) mice and wild-type (+/+, black symbols and bars) littermates were tested for two-bottle choice ethanol consumption and taste preference after the completion of the LORR test as described in Lasek et al. (2011b). (a) Amount of ethanol consumed (in g/kg/day) at 3%, 6%, 10%, 14% and 20% ethanol (v/v). (b) Ethanol preference expressed as a ratio based on total fluid consumption at the indicated concentrations of ethanol. (c) Amount of water consumed, expressed as g/kg/day. (d) Two-bottle choice taste test for sweet (saccharin, 0.03% and 0.06%) and bitter (quinine, 0.015 mM and 0.03 mM) solutions, expressed as a preference ratio based on total fluid consumption. No significant differences were found in any of the measures tested.
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