Alcohol intake and preference for C57BL/6J mice that consumed alcohol for 1, 2 or 4 weeks.

<p><b><i>A</i></b> The mice show a progressive increase in alcohol intake during the first two weeks of the experiment, stabilizing thereafter. <b><i>B</i></b> Preference for alcohol was high throughout the experiment. <b><i>C</i></b> RNA for microarray analysis was isolated from 0.6 mm diameter punches (<b>○</b>) from serial sections through the CeA. <b><i>D</i></b> The majority of the gene expression changes occurred after 1 week of alcohol consumption; the bar graph shows the total numbers of up- (black) and down-regulated (grey) genes for each time-point while the Venn Diagram shows the total number of up- and down-regulated genes (marked by arrows) for each time-point and overlap between the experimental groups. <b><i>E–F</i></b> Analysis of the microarray data using the <i>Short Time Series Expression Miner (STEM</i>), revealed significant gene enrichment particularly during the early stages of escalation of alcohol intake (week 1 and/or week 2). When considering all possible time profiles for gene expression after 1 wk, 2 wk and 4 wk of alcohol consumption, relative to data from naïve control mice (exemplified by <b><i>E</i></b>), 6 time profiles showed significant gene enrichment: they represented more genes than expected based on chance (<b><i>F</i></b>). The commonality between these 6 significant time profiles is that they all show an initial change in gene expression, normalizing thereafter to baseline levels.</p

Design and validation of 14-3-3ζ shRNA constructs

<p><b><i>A</i></b> 14-3-3ζ specific shRNA sequences (1222 and 1854 bp) were cloned into pLentiLox3.7 vectors and <b><i>B</i></b> western blot analysis revealed that both constructs effectively reduced 14-3-3ζ protein levels in Neuro2A cells, the 1854 construct being most effective <i>in vitro. </i><b><i>C</i></b><i> In situ</i> hybridization confirmed effective knockdown of 14-3-3ζ in the CeA after infection with the 1854 shRNA expressing lentivirus. The top panels show 14-3-3ζ expression in the CeA after infection with the control or the 1854 shRNA expressing lentivirus. The bottom panels show GFP mRNA and therefore the infection site in adjacent sections. 14-3-3ζ mRNA is completely absent in the area that is infected with the 1854 shRNA expressing lentivirus.</p

Taste control experiments show that the increase in alcohol intake in mice with CeA 14-3-3ζ knockdown is not secondary to altered taste sensitivity.

<p>Control mice and mice with CeA 14-3-3ζ knockdown showed equal intake of solutions containing <i>A</i> the caloric sweet tastant sucrose and <i>B</i> the non-caloric sweet tastant saccharin. <i>C</i> Aversion for the bitter tastant quinine was also not different between controls and mice with CeA 14-3-3ζ knockdown.</p

Significant effects of 1 week, 2 weeks and 4 weeks of daily alcohol consumption in the limited access choice paradigm on gene expression changes in the amygdala of C57BL/6J mice.

*<p>for the 1 week group only those genes are shown that display a change in expression of at least +/−20% from naïve controls. EtOH/N  =  fold change of the EtOH group (1 wk, 2 wk or 4 wk) to naïve controls. Hypothetical genes were excluded from this list.</p

Gene Enrichment analysis in STEM.

<p>Gene Ontology analysis for the significant time profiles 45 and 47 in STEM revealed significant enrichment of genes involved in transport, ion transport, ligand-gated ion channel activity, synaptic transmission, cytoplasm and protein transport. Highlighted are the selected <b>8 top candidate genes</b>.</p

qPCR data for mice that consumed alcohol for 1 week or 2 weeks.

<p>qPCR confirmed significant up-regulation of 4 out of 8 candidate genes when compared to naïve control mice: Gria3, 14-3-3 zeta, Gabrb3 and Prkacb. *P < 0.05, **P < 0.01 from naïve mice by Tukey HSD multiple comparisons.</p

Effects of local knockdown of 14-3-3ζ in the CeA on alcohol consumption. <i>A</i>

<p>Local knockdown of 14-3-3ζ in the CeA using the 1854 shRNA increased intake of a 10% alcohol solution (v/v). <b><i>B</i></b> Infection with the less effective 1222 shRNA also increased alcohol intake of a 10% alcohol solution (v/v), but less prominently so than the 1854 14-3-3ζ shRNA. <b><i>C</i></b> In a separate batch of mice, local knockdown of 14-3-3ζ in the CeA using the 1854 shRNA increased alcohol intake of a 15% alcohol solution (v/v) and <b><i>D</i></b> local knockdown of 14-3-3ζ in the CeA using the 1854 shRNA caused persistent preference for the alcohol solution despite adulteration with the bitter tastant quinine. In <b><i>E</i></b> the sites of viral infection in the brain are summarized. The black ellipses show the core of the infection site that was consistently targeted across all animals. The areas marked in grey represent less frequent infected sites that include the anterior amygdala, the basolateral amygdala and part of the caudate putamen, along the injection tract. • Control mice; <b>○</b> 14-3-3ζ-specific shRNA treated mice. * P<0.05 from controls; # P<0.05, ## P<0.01 from 0 μM quinine for mice treated with control lentivirus; $ P <0.05 from 0 μM quinine for mice treated with the 1854 14-3-3ζ shRNA expressing lentivirus by <i>t</i>-test.</p

Expression of <i>Th, NurrI</i>, <i>C130021L20Rik</i> and <i>Rspo2</i> is affected in the adult SNc.

<p>(A–G′′) <i>Th</i> expression in the adult midbrain from rostral to caudal, marking the SNc and the VTA respectively. A comparison between wild-type (wt), heterozygous and homozygous <i>Dreher</i> material demonstrates almost identical expression patterns between <i>wt/wt</i> and <i>wt/dr</i> coronal brain sections, whilst obvious defects can be seen in the rostral-lateral part of the SNc in <i>dr/dr</i> midbrains (arrowheads). The medial VTA displays some reduction, although more subtle than in rostral-lateral mdDA domains. (H–K′) Expression of <i>NurrI</i> (H,J,H′,J′), and <i>C130021L20Rik</i> (I,K,I′,K′), shows similar defects as was seen for <i>Th</i>; rostral-lateral expression is diminished (arrowheads). (L–Q′) A complete loss of <i>Rspo2</i> expression in the SNc was observed. In the wild-type, only a subset of cells in the SNc domain express <i>Rspo2</i>, and all <i>Rspo2</i> expression is lost in the affected SNc of the <i>Lmx1a</i> knock-out, as can be observed in higher magnifications (O,Q,O′,Q′).</p

<i>Lmx1a</i> Encodes a Rostral Set of Mesodiencephalic Dopaminergic Neurons Marked by the <i>Wnt</i>/B-Catenin Signaling Activator <i>R-spondin 2</i>

<div><p>Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor <i>Lmx1a</i> is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in <i>Lmx1a</i>-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of <i>Lmx1a</i>. Subsequent expression analysis on brain sections, confirmed that <i>Nurr1</i> is regulated by <i>Lmx1a,</i> and additional downstream targets were identified, like <i>Pou4f1, Pbx1, Pitx2</i>, <i>C130021l20Rik</i>, <i>Calb2</i> and <i>Rspo2</i>. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, <i>Nurr1</i> and <i>C130021l20Rik</i> were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the <i>Wnt</i>/b-catenin signaling activator <i>Rspo2</i> in this domain. Subsequent analysis of <i>Rspo2−/−</i> embryos revealed affected mdDA neurons, partially phenocopying the <i>Lmx1a</i> mutant. To conclude, our study revealed that <i>Lmx1a</i> is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the <i>Wnt</i> activator <i>Rspo2</i>.</p></div

mdDA markers TH, AHD2 and PITX3 display loss of expression in <i>Rspo2-LacZ/LacZ</i> embryonic midbrain.

<p>Sagittal sections of <i>Rspo2</i> control and knock-out (<i>LacZ/LacZ</i>) littermate mouse brains, at E14.5. (A–G′) TH protein expression analysis reveals a decrease in TH+ cells in the <i>Rspo2</i>-ablated mdDA neuronal field. Lines indicating the mid-hindbrain border (mhb) and the fasciculus retroflexus (fr) are added for clarity. (A′′–G′′) More detailed images of (A′–G′). BGAL protein staining can be observed in <i>Rspo2-LacZ/LacZ</i> mutant cells. Most of these cells do not express TH (anymore). (H–N′) A decrease in PITX3-expressing mdDA neurons in <i>Rspo2-LacZ/LacZ</i> tissue is shown. (O–U′) From lateral to medial to lateral, AHD2 analysis reveals a decrease in expression in the paramedian and lateral mdDA neuronal subset, in the absence of <i>Rspo2</i>. (V–X′) Higher magnifications of (C,C′), (J,J′) and (Q,Q′), showing a mild but clear decrease of mdDA neurons expressing TH, PITX3 and AHD2 in the paramedian midbrain. (Y–AA′) Higher magnifications of (F,F′), (M,M′) and (O,O′), showing a clear decrease in the number of TH-, PITX3-, and AHD2-positive neurons in the lateral mdDA neuronal field.</p