Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.
- Authors
- Bhandari, P; Hill, J S; Farris, S P; Costin, B; Martin, I; Chan, C-L; Alaimo, J T; Bettinger, J C; Davies, A G; Miles, M F; Grotewiel, M
- Year
- 2012
- Journal
- Genes, brain, and behavior
- PMID
- 22239914
- DOI
- 10.1111/j.1601-183X.2012.00765.x
- PMCID
- PMC3527839
Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol-related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)-Ξ² signaling, and regulation of ryanodine receptors and A-kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol-responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol-related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc-4 and exl-1, altered behavioral responses to acute ethanol in worms and that viral-mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.
Ethanol-responsive and basal expression of Clic4 in mouse PFC(A) qRT-PCR analysis of basal (Saline) and ethanol-responsive (EtOH, 4 g/kg, 4 hr) Clic4 expression in DBA2/2J mice. Expression of Clic4 is elevated after ethanol treatment, validating prior microarray results (Kerns et al., 2005) . (B and C) Pearson correlation of Clic4 basal expression in PFC (x-axis) of BXD recombinant inbred lines (numbered points) with ethanol-induced locomotor activity (GeneNetwork trait ID 11962 (Philip et al., 2010)) (B) and initial sensitivity to ethanol-induced rotarod ataxia following first of five injections (onset of ataxia brain ethanol threshold, mg ethanol/g brain β GeneNetwork ID 10144 (Gallaher et al., 1996)) (C). Scattergrams were generated in GeneNetwork (www.genenetwork.org) using the VCU PFC saline database (Wolen and Miles, unpublished). B6 and D2 strains were not tested in experiments shown in panel B
Transposon insertions cause partial loss of function in Drosophila Clic(A) The Clic locus and transposon insertions. Transcription of Clic is from left to right. The Clic transcription unit is represented by the filled rectangle with nucleotide positions indicated above (coordinates from FlyBase annotation release 5.33). Exons are represented by rectangles below the transcription unit with protein coding sequences and untranslated regions depicted as grey and open rectangles, respectively. Introns are represented as a line and transposons as triangles. Scale bar (upper right) is 1000 bp. Schematic adapted from FlyBase. (B) Whole-body Clic mRNA expression in transposon lines. Expression of Clic mRNA in flies heterozygous for the G0472 and the EY04209 transposons was reduced relative to w1118 controls.
Ethanol sensitivity in Drosophila Clic transposon mutants and revertantsEthanol sensitivity represented as T50 values in ClicG0472/+ and ClicEY04209/+ (black bars, A and B, respectively) and Control w1118 flies (open bars). Clic mutants had significantly higher T50 values than controls. (C) Ethanol sensitivity (T50 values) in ClicG0472 heterozygous transposon mutants and revertants. ClicG0472/+ flies (black bar) had higher T50 values than Control w1118 flies or revertants (ClicG0472.R4 and ClicG0472.R9) (white bars).
Internal ethanol concentrations in Drosophila Clic mutantsClicG0472/+ (A) and ClicEY04209/+ (B) were exposed to ethanol vapor for the indicated durations in eRING assays in parallel with Control w1118 flies. Internal ethanol concentrations were determined as described in Materials and Methods
Ethanol sensitivity and acute functional tolerance in C. elegans with mutations in Clic orthologues(A) Effect of ethanol exposure on relative locomotor speed (percent of untreated animals) in N2 control (open circles) and Clic mutants (exl-1(ok857), light grey squares; exc-4(rh133), dark grey triangles; exc-4(rh133);exl-1(ok857), black diamonds). Data are from 4 independent experiments where 10 worms per genotype contributed to an average speed for a population. (B) Internal ethanol concentrations in N2 control and Clic mutants were determined as described in Materials and Methods
Altered loss of righting reflex (LORR) in mice with AAV2 viral vector-mediated expression of Clic4 in brainAAV2 vectors expressing a Clic4-FLAG fusion protein (AAV-CLIC4; panel A) or empty vector (AAV-IRES; panel B) were stereotactically injected into male DBA/2J mouse PFC. Panels A and B show immunohistochemistry results for FLAG epitope primary antibody staining. Immunohistochemistry was done 2 weeks after the last behavioral studies (~9 weeks after viral injections). Panel C shows that Clic4 over-expressing animals (black) had a shorter duration of LORR following 3.8 g/kg IP of ethanol compared to control (grey). Behavioral testing for LORR was done ~7 weeks after viral injections.
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