A Drosophila model for alcohol reward. Supplemental Material

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1 Kaun et al. Supplemental Material 1 A Drosophila model for alcohol reward Supplemental Material Kaun, K.R. *, Azanchi, R. *, Maung, Z. *, Hirsh, J. and Heberlein, U. * * Department of Anatomy, University of California, San Francisco, CA Department of Biology, University of Virginia, Charlottesville, VA 22904, USA Supplementary Table 1: Odor controls for UAS/GAL4 experiments. p. 2 Supplementary Table 2: Ethanol controls..... p. 3 Supplementary Table 3: Statistics for Manuscript figures p. 5 Supplementary Table 4: Statistics for Supplementary figures..... p. 6 Supplementary Table 5: Odor controls for sca lines..... p. 4 Supplementary Figure 1....p. 7 Supplementary Figure 2....p. 8 Supplementary Figure 3.p. 9 Supplementary Figure 4.p. 10 Supplementary Figure 5.p. 11 Supplementary Figure 6.p. 12 Supplementary Figure 7.p. 13

2 Kaun et al. Supplemental Material 2

3 Kaun et al. Supplemental Material 3

4 Kaun et al. Supplemental Material 4

5 Kaun et al. Supplemental Material 5

6 Kaun et al. Supplemental Material 6

7 Kaun et al. Supplemental Material 7 Supplementary Figure 1: Conditioned aversion to preference for ethanol (a) Apparatus for conditioned preference for an odor associated with ethanol in Drosophila. One of two odors was paired with vaporized ethanol in training chambers, then flies chose between the unpaired and paired odors in a Y-maze (see methods in Supplementary Material for details). (b) When naïve flies were given a choice between the two odors used, they showed no inherent preference (p=0.52) (c) The switch from conditioned aversion to conditioned preference occurs about 12 hrs post-training (Tukey s post-hoc 0.5hr-12 hr Group A p<0.05, 15hr-24 hr, Group B p<0.05).

8 Kaun et al. Supplemental Material 8 Supplementary Figure 2: Conditioned response requires a cue-ethanol association (a) Sham training with three spaced sessions of odor alone or 53% ethanol alone did not result in conditioned aversion 30 min after training (odor p=0.08, ethanol p=0.69) or (b) conditioned preference 24 hrs after training (odor p=0.69, ethanol p=1.00). (c,d) A protocol where ethanol is unpaired from odors fails to induce either conditioned aversion (p=1.00) or preference (p=0.40) suggesting that the conditioned responses to the odors are contingent on the paired presentation of odors and ethanol.

9 Kaun et al. Supplemental Material 9 Supplementary Figure 3: Flies will endure a stronger shock for ethanol than sugar (a) Flies trained to associate an odor with an ethanol reward show a CPI of over 0.2, which translates into about 60% of flies willing to walk over an inactive electric grid to obtain the odor associated with ethanol. (b) Flies trained to associate an odor with a 2M sucrose reward show a Learning Index of over 0.3 which translates into about 65% of flies willing to walk over an inactive electric grid to obtain the odor associated with sucrose. (c) Ethanol absorption increases as the dose of vaporized ethanol increases.

10 Kaun et al. Supplemental Material 10 Supplementary Figure 4: Dopamine is required for conditioned preference (a) At the permissive temperature when neurotransmission was not blocked, 24 C, shi ts1 in THand Ddc- expressing cells did not have an affect on conditioned aversion or (b) conditioned preference. (c) HPLC of dissected brain tissue revealed that 10 mg/ml 3IY significantly and specifically decreased brain levels of dopamine and (d) 20mM αmtp significantly and specifically decreased brain levels of serotonin.

11 Kaun et al. Supplemental Material 11 Supplementary Figure 5: The mushroom body is required for conditioned preference (a) We transiently inactivated neurotransmission in select sets of mushroom body neurons using the GAL4 drivers OK107, 201Y, MB247, 5-66a and OK107, 201Y and MB247 have been previously characterized. 5-66a and 4-59 were isolated from a collection of P{GawB} enhancer traps. This summary roughly represents the expression of these drivers in different subsets of mushroom body neurons. (b)-(f) expression of mushroom body GAL4 drivers in the adult male central brain: GAL4-GFP = green, nc82 neuropil marker = magenta (b) OK107 (c) MB247 (d) 201Y (e) 5-66a (f) 4-59 (g) At the permissive temperature, shi ts expressed in subsets of the mushroom body did not have an effect on conditioned aversion or (h) preference with the exception of OK107 (30 min p=1.00, 24 hrs p=0.50).

12 Kaun et al. Supplemental Material 12 Supplementary Figure 6: sca mutants do not have morphological defects in the mushroom body. All flies used in behavior and all brains dissected for analysis were from adult males. (a) nc82 staining in a representative wild-type (Berlin) control central brain. (b) nc82 staining in a representative sca homozygote central brain. (c) fasii staining in a representative wild-type (Berlin) control central brain. (d) fasii staining in a representative sca homozygote central brain. No gross morphological differences were observed between wild-type and sca mutant mushroom body αβ neurons visualized with fasii. (e) A trace of the mushroom body from a representative wild-type (Berlin) control brain stained with nc82. (f) A trace of the mushroom body from a representative sca homozygote brain stained with nc82. No gross morphological differences were observed in the entire mushroom body neuropil structure between wildtype and sca mutants.

13 Kaun et al. Supplemental Material 13 Supplementary Figure 7: expression in the adult male central brain GFP = green, nc82 neuropil marker = magenta. (a) expression in the central brain. (b) is expressed in a number of cell bodies clustered around the ventrolateral protocerebrum and subesophogeal ganglia. The areas that the neuronal processes innervate are not characterized here. (c) is expressed in neurons in the lobular plate and eye. (d) is expressed in a number of glomeruli in the antennal lobe. (e) Kenyon Cells expression in the calyx. (f) is expressed in the mushroom body αβ neurons. No expression in the α β neurons was observed. (g) is expressed in the γ neurons.