Figure 1.
Ethanol preference is based on olfactory information. a
Flies are offered a choice between two odor traps and flies will normally decided for one or the other trap within 16 h. The preference index (PI) indicates the percentage of flies that prefer one odor over the other. A positive PI is defined as preference and a negative PI indicates aversion. b Control and heterozygote Orco1 mutants prefer ethanol containing food odors, whereas transheterozygote Orco1/Orco2 and homozygote Orco1 mutants do not show preference (PIs are w1118 0.19±0.07, Orco1/+0.55±0.05, Orco1/Orco2 −0.01±0.07 and Orco1/Orco1 0.11±0.08. n = 24, 26, 32 and 31. P<0.01 and P<0.001). c Flies prefer food odors over water (PIs are w1118 0.91±0.02 and w1118; Orco1 0.77±0.03. n = 29. There is a significant difference for food preference between w1118 and Orco1. P<0.05) and ethanol over water (PIs are w1118 0.76±0.06 and w1118;Orco1 0.8±0.04. n 11 and 13.). d Grey bars represent the choice between food odor and ethanol (PIs are w1118 0.69±0.04 and w1118;Orco1 0.52±0.05. n 33.) and dark grey indicates the choice between food odor with ethanol versus ethanol alone (PI are w1118 0.87±0.03 and w1118;Orco1 0.52±0.06. n = 29 and 30. The olfactory preferences of w1118 for different tested conditions differ significantly. P<0.05). Bars labeled with a are significantly different from random choice as determined by One-sample sign test.
Figure 2.
Ethanol preference depends on TbH function.
a TbhnM18 mutants do not show preference and differ significantly from controls (PIs are TbhnM18 0.09±0.09 and control 0.23±0.08. n 21 and 23. P<0.01). The loss of preference is independent of w1118 (PIs are w1118 0.31±0.06 and w1118, TbhnM18 0.08±0.09. n 26 and 17. P<0.05 and P<0.01). b Tdc2-GAL4 dependent expression of TbH in TbhnM18 mutants restores preference to control levels (PIs are w1118;Tdc2-GAL4/+0.34±0.05, w1118; UAS-TbH 0.36±0.07, w1118,TbhnM18;Tdc2-GAL4/+ −0.14±0.08, w1118,TbhnM18;UAS-TbH/+ −0.01±0.08 and w1118,TbhnM18;Tdc2-GAL4/UAS-TbH 0.34±0.1. n 21, 28, 23, 21 and 21.). Mutants carrying the transgenes insertions in the mutant background differ significantly from control carrying the respective transgene (P<0.05 and P<0.01). c TbhnM18 mutants prefer food odor over water similar to the controls (PIs are for w1118 0.77±0.03 and w1118, TbhnM18 0.81±0.03. n 18 and 15.) but do not prefer ethanol odor over water to the same extend as the controls (PIs are w1118 0.67±0.05 and w1118,TbhnM18 0.31±0.08. n 20 and 21. P<0.001). d Control flies prefer complex odors over the single odor ethanol (PI is w1118 0.36±0.06. n 35.) and increase their preference significantly for the complex odor when ethanol is added (PI is w1118 0.81±0.03. n 18, P<0.001). TbhnM18 flies show an increased preference for complex odors with ethanol over ethanol in comparison to the choice of complex odors without ethanol and ethanol (w1118,TbhnM18 0.10±0.08 and 0.74±0.04. n 24 and n 17. P<0.001). The letter a marks differences from random choice as determined by a One-sample sign test.
Figure 3.
The Tdc2-GAL4 activates UAS transgenes in a subset of TbH positive neurons.
The GAL4 expression domain of the Tdc2-GAL4line is visualized by GFP in comparison to TbH expression in the anterior (a) and posterior (b) part of the adult brain. Neurons expressing TbH (G0a) or GFP (G5a) and neurons expressing both (VMI) are highlighted with an arrow (a). In the back of the brain G4a neurons only express TbH, whereas VMII and VMIII neurons express both (b). The expression of Tbh (c to f) and GFP (c′ to f′) is found in the same set of neurons of the G3a/AL2 cluster (c′′), VMI (d′′), VMII (e′′) and VMIII (f′). The analysis of expression patterns is summarized in table g and clusters in which GFP and Tbh co-localize are highlighted in grey. The scale bar represents 25 µm.
Figure 4.
TbH is required in a subset of octopamingergic neurons for ethanol preference.
a Expression of TbH in a Tdc2-GAL4; Cha-GAL80 dependent manner in TbhnM18 mutants does not restore preference (PIs are Tdc2-GAL4/+; Cha-GAL80/+: 0.26±0.05, TbhnM18; Tdc2-GAL4; Cha-GAL80/+0.23±0.08 and TbhnM18, UAS-Tbh; Tdc2-GAL4; Cha-GAL80/+0.00±0.10, n 27, 22 and 25. The experimental group does not develop preference and differs significantly from the control. The transgene insertion in the mutant background differs significantly from respective control. P<0.05 and <0.001.). b Feb15-GAL4 dependent expression of TbH in TbhnM18 mutants restores preference (PIs are w1118; Feb15-GAL4/+0.37±0.07; w1118, TbhnM18; Feb15-GAL4/+0.14±0.07 and w1118, TbhnM18, UAS-Tbh; Feb15-GAL4/+0,47±0.04. n 28, 33 and 36. The level of preference of the experimental group does not differ from the control. The transgene insertion in the mutant background differs significantly from respective control. P<0.05.). c Loss of preference in TbhnM18 mutants is not completely restored by TbH expression under the control of the 6.2-Tbh-GAL4 driver (PIs are w1118; 6.2-Tbh-GAL4/+0.29±0.06, w1118, TbhnM18; 6.2-Tbh-GAL4/+0.06±0.08 and w1118, TbhnM18, UAS-Tbh; 6.2-Tbh-GAL4/+0.13±0.08. n 36, 26 and 36. The olfactory preferences of the control group and experimental group are not significantly different. Mutants carrying one copy of the GAL4 transgene differ significantly from the control carrying one copy of the GAL4 transgene. P<0.05.). The schemata below graphs for behavioral experiments summarize the expression domains of respective GAL4 drivers in comparison to TbH expression. White cells are TbH positive neurons not targeted by specific driver lines and grey cells indicate co-localization of GAL4 and TbH. The letter a labels an olfactory preference score that is significantly different from random choice as determined by One-sample sign test.
Figure 5.
Neuronal activity is required for preference.
After activation of UAS-Kir2.1 under the control of Tdc2-GAL4, tub-GAL80ts preference is significantly reduced (PIs are Tdc2-GAL4, tub-GAL80ts; UAS-Kir2.1 0.4±0.1 and with HS 0.12±0.09. n 22. P<0.05). Activation of a UAS-mCD8::GFP construct does not interfere with preference (PIs are Tdc2-GAL4, tub-GAL80ts; UAS-mCD8::GFP 0.22±0.16 and with HS 0.54±0.17. n 14.). Transgene insertions do not alter preference of flies carrying UAS-Kir2.2 and tub-GAL80ts (PIs are w1118; tub-GAL80ts; UAS-Kir2.1 0.29±0.11 and with HS 0.37±0.09. n 27 and 28.).
Figure 6.
Neuronal activity is sufficient to induce preference. a
The behavioral set up of the optogentic trap assay consists of two odor traps filled with food odor surrounded by a dark intransparent plastic. The assay is placed on a cold light plate to motivate flies to descend into the fly traps. On top of the two odor traps two different diodes – one for blue light and one for yellow – are placed that can be activated with the different frequencies. b Light activation of neurons in a Tdc2-GAL4 dependent manner causes preference for the trap illuminated in blue (PIs are norpA1, Tdc2-GAL4/UAS-ChR2;UAS-ChR2 with vehicle −0.02±0.11 and with retinal 0.35±0.09; n 12 for both. P<0.05.). c Typical traces of norpA1,Tdc2-GAL4/UAS-ChR2;UAS-ChR2 and norpA1,TH-GAL4/UAS-ChR2;UAS-ChR2 flies before and after a one min illumination period are shown. The average activity of 7 flies per genotype and condition are summarized in the table. d Activation of dopaminergic neurons causes aversion for the trap illuminated in blue (PIs are norpA1,TH-GAL4/UAS-ChR2;UAS-ChR2 with vehicle −0.07±0.09 and with retinal −0.41±0.1. n 20 and 35. P<0.05.).