1. Volume 70, Issue 5, Pages 979-990 (June 2011)
arouser Reveals a Role for Synapse Number in the Regulation of Ethanol Sensitivity 
Mark Eddison, Douglas J. Guarnieri, Ling Cheng, Che-Hsiung Liu, Kevin G. Moffat, Graeme Davis, Ulrike Heberlein 
Neuron 
Volume 70, Issue 5, Pages (June 2011)
DOI: /j.neuron
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
arouser Mutants Show Increased Sensitivity to Ethanol Sedation
(A) aru8.128 flies had increased sensitivity to ethanol-induced loss of postural control in the inebriometer. Ethanol sensitivity was measured as the mean elution time (MET). The aru8.128 mutant had a significantly decreased MET compared to control flies (∗∗∗p < 0.001; n = 11–14; unpaired t test).
(B) The ethanol sedation curve of aru8.128 and aru8896 flies in the loss-of-righting-reflex (LORR) assay, both showing increased sensitivity compared to control. Ethanol exposure commenced at 0 min and was continuous throughout the experiment (dose = 110/40 Ethanol/Air [E/A]). Significantly increased ethanol sensitivity was observed for both aru mutants at the 25, 50 and 75 percentiles (∗∗∗p < 0.001; n = 9).
(C) aru8.128 and aru8896 both had decreased median sedation time in the LORR assay. The median sedation time (ST50) is the time required for half of the ethanol-exposed flies to show a LORR. ST50 was calculated by linear interpolation. Significant differences were observed between control and aru8.128 or aru8896 (∗∗∗p < 0.001; n = 9).
(D) Precise excision of the P element in aru8.128 (aruΔ8.128) restored normal ethanol sensitivity. Statistical significance was observed between aru8.128 and aruΔ8.128 (∗∗∗p < 0.001), but not between control and aruΔ8.128 (p > 0.05) (n = 8).
In this and all other figures, data are represented as mean ± SEM and, unless stated, all statistical tests were one-way ANOVAs with Newman-Keuls post-hoc test.
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3. Figure 2 Molecular Characterization of the aru Locus and aru Mutants
(A) Schematic representation of the aru transcription unit. Gray boxes represent UTRs and black boxes represent coding regions. Structures of two transcripts, aru-RA and aru-RD, and their respective transcription start sites, T1 and T2, are shown, as well as insertion sites of aru8.128 and aru8896. The aru-RA product is predicted to include an additional 27 amino acids at the N terminus (represented as a white box), which is noncoding in aru-RD. Black bars represent the aru common or aru-RD-specific probes used in northern-blot analysis. aruRNAi and aruRNAi-2 define the regions targeted by RNAi constructs.
(B–D) Expression of aru-RD is prevented by the aru8.128 mutation.
(B) Northern blot on mRNA isolated from adult flies using a probe that recognizes both aru-RA and aru-RD showed a reduction in band intensity (at ∼ 3 Kb) in aru We did not detect a 3 Kb transcript in aru8.128 mutant with an aru-RD specific probe, but did in a precise excision line aruΔ A tubulin84B probe was used to control for total mRNA levels.
(C) RT-PCR of aru-RD at all developmental stages. aru-RD was expressed throughout the life of the fly, but was not detected at any stage in aru8.128 flies.
(D) Western-blot analysis of adult heads or bodies revealed the aru8.128 mutation results in a lack of Aru expression in the head and a reduction in the body.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3
aru Acts in Neurons during Development to Reduce Ethanol Sensitivity
(A) Aru is expressed in neurons. Western blot of adult head extracts of elavc155-GAL4;UAS-aruRNAi/+ flies shows a strong reduction of Aru expression. A tubulin antibody was used as a loading control.
(B) As measured by the ST50, silencing aru expression in neurons by driving UAS-aruRNAi with elav-GAL4 c155 increased ethanol sensitivity (∗∗∗p < 0.001; n = 8).
(C) Expression of UAS-aruRNAi (29°C) until eclosion (star) using elav-GAL4c155;GAL80ts and then switching off expression (18°C) in the adult fly for 4 days prior to behavioral testing (arrow) was sufficient to increase ethanol sensitivity (∗∗∗p < 0.001; n = 11).
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5. Figure 4
aru Is Required for Egfr/Erk Pathway Regulated Decrease in Ethanol Sensitivity
(A) Overexpression of UAS-rlact with elavc155-GAL4 decreased ethanol sensitivity, but only in the presence of Aru. Significance was observed between elav-GAL4c155 and elav-GAL4c155;UAS-rlact/+ (p∗∗∗ < 0.001), and between elav-GAL4c155 and elavc155-GAL4;aru8.128 (∗∗p < 0.01), but not between elavc155-GAL4;aru8.128 and elavc155-GAL4;aru8.128;UAS-rlact/+ (p > 0.05) (n = 8).
(B) Compared to control flies, the partial loss-of-function hppy17.51 mutant flies had decreased ethanol sensitivity (∗∗∗p < 0.001) and aru8.128 had increased ethanol sensitivity (∗∗∗p < 0.001). The double mutant aru8.128;hppy17.51 had increased ethanol sensitivity (∗∗∗p < 0.001) not significantly different than aru8.128 (p > 0.05) (n = 8).
(C) A model of aru function regulated by the Egfr/Erk pathway. The Egfr/Erk pathway and aru both function to reduce ethanol sensitivity. aru functions downstream of hppy and rl/Erk. hppy functions as a negative regulator of Egfr signaling, upstream of rl/Erk (Corl et al., 2009).
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6. Figure 5
Activation or Inhibition of PI3K/Akt Signaling in Neurons Alters Ethanol Sensitivity
(A–F) Flies in which various genes in the PI3K/Akt pathway were manipulated under the control of elavc155-GAL4 were measured in the LORR assay. Flies with neuronal overexpression of (A) UAS-PI3K92E, (D) PDK1, and (E) UAS-Akt1 showed increased ethanol sensitivity. Flies with neuronal overexpression of (B) UAS-PI3KDN, (C) UAS-Pten, (F) UAS-AktRNAi showed decreased ethanol sensitivity (∗∗∗p < 0.001) (∗∗p < 0.01 for elavc155-GAL4/UAS-PI3K92E versus UAS-PI3K92E) (n = 8).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
aru Is Required for PI3K/Akt Pathway Regulated Decrease in Ethanol Sensitivity
(A) Overexpression of PI3KDN with elavc155-GAL4 decreased ethanol sensitivity, but only in the presence of Aru. Significance was observed between elavc155-GAL4 and elavc155-GAL4; UAS-PI3KDN/+ (p∗∗∗ < 0.001) and between elavc155-GAL4 and elavc155-GAL4;aru8.128 (p∗∗ < 0.01), but not between elavc155-GAL4;aru8.128 and elavc155-GAL4;aru8.128;UAS PI3KDN/+ (p > 0.05) (n = 10).
(B) Compared to control flies, the partial loss-of-function Akt mutant, Akt1EY1002/+ (lethal as a homozygote), had decreased ethanol sensitivity (∗∗∗p < 0.001) and aru8.128 had increased ethanol sensitivity (∗∗∗p < 0.001). The double mutant aru8.128;Akt1EY1002/+ had increased ethanol sensitivity (p∗∗∗ < 0.001) not significantly different from aru8.128 (p > 0.05) (n = 8).
(C) A model of aru function regulated by the PI3K/Akt pathway. The PI3K/Akt pathway enhances ethanol sensitivity by inhibition of aru, which functions to reduce ethanol sensitivity.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
The Egfr/Erk and PI3K/Akt Pathways Have Different Temporal Requirements
(A–D) Temporary neuronal overexpression of Egfr or rlact did not decrease ethanol sensitivity.
(A and B) Overexpression of (A) UAS-Egfr or (B) UAS-rlact with elav-GAL4;GAL80ts only until eclosion (star) and switching it off in the adult fly for 4 days prior to behavioral testing (arrow) did not decrease ethanol sensitivity. No significant difference was observed between elavc155-GAL4;GAL80ts/+ and elavc155-GAL4;GAL80ts/+;UAS-Egfr/+ or elavc155-GAL4;GAL80ts/+;UAS-rlact (p > 0.05), but was observed between GAL80ts/+;UAS-Egfr/+ and elavc155-GAL4;GAL80ts/+;UAS-Egfr/+ (∗∗p < 0.01) and GAL80ts/+;UAS-rlact and elavc155-GAL4;GAL80ts/+;UAS-rlact/+ (∗p < 0.05) (n = 8). Significance was only attributed to the experimental line if statistically different from both GAL4/+ and UAS/+ controls. This experiment was conducted at 27°C because overexpression of Egfr and rlact at 29°C was lethal, while raising flies at 27°C restored viability and health, and sufficiently turned off GAL80ts (see Figures S5A–S5D).
(C and D) Using elavc155-GAL4;GAL80ts to overexpress UAS-Egfr (C) or UAS-rlact (D) only after eclosion (star) for 3 days in the adult fly prior to behavioral testing (arrow) did not decrease ethanol sensitivity (p > 0.05; n = 8).
(E–H) The PI3K/Akt pathway is required during development to alter ethanol sensitivity.
(E and F) Overexpression of (E) UAS-PI3KDN or (F) UAS-Akt1 with elavc155-GAL4;GAL80ts only until eclosion (star) and switching off in the adult fly for 4 days prior to behavioral testing (arrow) decreased and increased ethanol sensitivity, respectively (∗∗∗p < 0.001; n = 8).
(G and H) Overexpression of (G) UAS-PI3KDN or (H) UAS-Akt1 with elavc155-GAL4;GAL80ts only after eclosion (star) for 3 days in the adult fly prior to behavioral testing (arrow) did not alter ethanol sensitivity (p > 0.05; n = 8).
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions

9. Figure 8
aru8.128 Flies Have Increased Synaptic Terminals in Adult PDF Neurons That Are Normalized By Social Isolation
(A) Silencing aru expression in the PDF neurons by driving UAS-aruRNAi with Pdf-GAL4 increased ethanol sensitivity (∗∗p < 0.01; n = 8).
(B and C) Confocal images of adult PDF neurons in (B) control and (C) aru8.128 flies, both expressing UAS-syt-GFP with Pdf-GAL4 and stained with an antibody against GFP.
(D) Quantification of synaptic terminals shows a significant increase in PDF neurons of aru8.128 flies compared to control (p∗∗∗ < 0.001; n = 8–10; unpaired t test).
(E) Adult social isolation of control flies significantly reduces ethanol sensitivity (∗∗∗p < 0.001; n = 6; unpaired t test).
(F) Adult social isolation of aru8.128 and aru8896 flies restored ethanol sensitivity to the level of control grouped flies (p > 0.05; n = 8).
(G) Adult social isolation normalized synapse number in aru8.128 PDF neurons to the level of control grouped flies (p > 0.05; n = 8–10).
Neuron  , DOI: ( /j.neuron )
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10. Figure 9 aru Regulation of Acute Ethanol Sensitivity
A model of aru regulation of ethanol sensitivity in neurons. aru is a negative regulator of ethanol sensitivity that genetically interacts with two pathways. In the Egfr/Erk pathway, Erk signaling promotes aru, which reduces ethanol sensitivity through an unknown mechanism. In the PI3K/Akt pathway, Akt signaling inhibits aru, which enhances ethanol sensitivity by its negative control of synapse number. Rheb, which functions in an independent pathway regulated by Tsc1/2, also increases synapse number and ethanol sensitivity. Conversely, adult social isolation decreases synapse number and ethanol sensitivity.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions