D. melanogaster genotypes. Canton-S wild-type D. melanogaster larvae or adults were used for all experiments, unless mentioned otherwise. Flies carrying P(acman) brp83 have been described previously (58), and because they carry two additional copies of the gene coding for BRP, these animals are referred to as “4×BRP” for simplicity. The “2×BRP, baseline” genotype, having only the genomic copies of BRP-coding genes, corresponds to the Canton-S strain mentioned above.

Food media and fly keeping. Procedures followed those of (59). To prepare standard food medium, 34 liters of water were mixed to 5.9 kg of cornmeal (Mühle Hofmann, Röthlein, Germany), boiled for 5 min, and automatically stirred gently for 4 hours. On the next day, 400 g of soya flour (Mühle Hofmann, Röthlein, Germany), 750 g of dried yeast powder (Heirler Cenovis, Radolfzell, Germany), and 250 g of agar-agar (Carl Roth, Karlsruhe, Germany) were added to 6 liters of water; after stirring, 1.8 liters of malt (Ulmer Spatz, Bingen am Rhein, Germany) and 1.8 liters of sugar beet molasses (Grafschafter Krautfabrik, Meckenheim, Germany) were added and boiled for 5 min with the cornmeal mixture while being gently stirred. Upon cooling to 70° to 80°C, 100 g of antifungal agent (methyl-4-hydroxybenzoate; Merck, Darmstadt, Germany) was added.

To prepare fly culture vials, this food medium was boiled in a microwave oven, and, for control vials, aliquots (20 ml) were poured into plastic vials and kept at 4°C for later use. For the experimental groups, each of the following substances was added 5 min after boiling to reach the specified concentrations of material per volume of food. These vials were stored for later use at 4°C

(1) Rhodiola1: Dried R. rosea roots (collected by O.L. in 2009 in the Carpathian Mountains near Mount Pip Ivan; 48°2′31″N, 24°37′32″E) were ground for 60 s with a commercial coffee mill. The powder was added to the vials 5 min after boiling to reach the specified concentrations; then, vials were stored at 4°C.

(2) Tablet: Ground “Arctic root” tablets (Swedish Herbal Institute, Gothenburg, Sweden; lot no. 60363, purchased via s.a.m. Pharma, Vienna, Austria; expiration date: November 2011, implying harvest of plant material before that date) were added to the food to reach the indicated concentrations. According to the manufacturer’s specifications, 28% of the tablets’ weight consists of the patented SHR-5 extract of R. rosea. Assuming that this extract is enriched from the dried root ingredients by at least a factor of 10, a concentration of 2.8 mg/ml of tablet should thus correspond to ~10 mg/ml of Rhodiola1; a pilot experiment had shown that higher concentrations of the hygroscopic ground tablet powder compromised the viability of Drosophila.

(3) Rhodiola2 refers to a second crop of dried R. rosea roots, also collected by O.L. in 2009 in the Carpathian Mountains near Mount Pip Ivan (48°2′31″N, 24°37′32″E).

(4) Rhodiola3 refers to dried R. rosea roots of unspecified Russian origin purchased by O.L. before 2011 and thus also harvested before that date.

(5) Rhodiola4 root was purchased by B.M. in 2011 from the Eveline24.de online shop (Maardu, Estonia; Ch./lot no. 18621; expiration date: 6 July 2014 and thus harvested before that date).

Voucher samples of all accessions or purchases are deposited at the Leibniz Institute of Plant Biochemistry and the Leibniz Institute for Neurobiology: Arctic root tablets (QGB005), Rhodiola1 (QGB001), Rhodiola2 (QGB003), Rhodiola3 (QGB004), and Rhodiola4 (QGB011).

In all cases, vials were retrieved from the 4°C store at around noon, and 2 hours afterward, approximately 100 Canton-S wild-type flies were added to the vial, which was then maintained at 25°C and 60 to 70% relative humidity under a 14-hour light/10-hour dark cycle. On the next day, these flies were removed; after an additional 4 days, the larvae were harvested from the food slurry for experiments.

Preparation of Rhodiola extractcrude for food medium. Finely chopped Rhodiola4 root (1.565 kg) was exhaustively extracted with 80% aqueous undenatured ethanol (3 × 6 liters) at room temperature. The extracts were combined and filtered, and the solvent was evaporated under reduced pressure. Food medium was then prepared as described above, at the concentrations mentioned in Results.

Synthesis of ferulic acid esters and derivatives for food medium. The ferulic acid esters FAE-8, FAE-12, FAE-16, and FAE-20 were synthesized by the Mitsunobu reaction from FA and long-chain alcohols as described by Maresca et al. (60). In the same fashion, 4-OH-CAE-20 was obtained starting from trans-p-coumaric acid, and icosanol. DH-FAE-20 was obtained by hydrogenation of the double bond of FA eicosyl ester, affording the product in quantitative yield. Synthesis details and compound data are summarized in Supplementary Materials and Methods. Food medium was then prepared as described above, at the concentrations mentioned in Results.

Synthesis of BSSG and derivatives for food medium. The synthesis of β-sitosterol-β-d-glucoside and β-sitosterol-β-d-galactoside was performed following a procedure described by Kunz and Harreus (61), starting from acetobromo-α-d-glucose or acetobromo-α-d-galactose and sitosterol. The remaining compounds were purchased from commercial sources: β-sitosterol (Honeywell-Fluka via Fisher Scientific, Schwerte, Germany), stigmasterol (Acros Organics, Geel, Belgium), stigmasterol-β-d-glucoside (ChemFaces, Wuhan, Hubei, China), and cholesterol-β-d-glucoside and stevioside (both from Sigma-Aldrich, Taufkirchen, Germany). Food medium was then prepared as described above, at the concentrations mentioned in Results.

Behavioral experiments in larval Drosophila. The procedures for behavioral experiments in larval Drosophila follow those of (59) and are further specified below.

Two-odor learning paradigm in larval Drosophila. The learning experiments follow the procedures of (62) (see sketch in Fig. 1A): Petri dishes (Sarstedt, Nümbrecht, Germany) with an inner diameter of 85 mm were filled with 1% agarose (electrophoresis grade; Carl Roth, Karlsruhe, Germany), which was allowed to solidify. The dishes were covered with their lids and then left untreated at room temperature until the following day. As the sugar reward, 2 mol of FRU (purity: 99%, Carl Roth, Karlsruhe, Germany) was used, which was added to 1 liter of agarose 10 min after boiling.

Experiments were performed under natural light at 21° to 24°C. Before the experiments, the regular lids of the petri dishes were replaced by lids perforated in the center by 15 holes (diameter, 1 mm) to improve aeration.

Odor was applied by adding 10 μl of odor substance into custom-made Teflon containers (inner diameter, 5 mm; these could be closed by a perforated lid with seven holes, 0.5 mm in diameter each). As odors, we used AM [Chemical Abstracts Service (CAS) no. 628-63-7; purity, 98.5%, diluted 1:50 in paraffin oil] and 1OCT [CAS no. 111-87-5; purity, 99% (undiluted)], both from Merck (Darmstadt, Germany), unless stated otherwise.

A spoonful of food medium containing larvae was taken from the food vial and transferred to a droplet of tap water on a petri dish. Thirty animals were collected, briefly washed in tap water, and transferred as a group to the assay plates for the start of training; in half of the cases, we started with a FRU-containing petri dish, and in the other half of the cases, we started with an agarose-only (PURE)–containing petri dish.

Immediately before the first training trial, two containers both loaded with the same odor were placed onto the assay plate on opposite sides of the plate (7 mm from the edges). Within each reciprocal training condition, we started with AM in half of the cases and with 1OCT in the other half, unless stated otherwise. Then, the petri dish was closed, and the larvae were allowed to move freely for 5 min. The larvae were then transferred to a petri dish with the alternative odor and the respective other substrate for 5 min (e.g., AM was presented on a FRU-containing plate and 1OCT on a PURE petri dish: AM+/1OCT training). This cycle was repeated two more times. Fresh petri dishes were used for each trial.

After this training, the animals were tested for their choice between the odors. The larvae were placed in the middle of a PURE petri dish; unless mentioned otherwise, a container with AM was placed on one side and a container with 1OCT was placed on the other side to create a choice situation. After 3 min, the number of animals on the “AM” or “1OCT” side was counted. After this test was completed, the next group of animals was run and trained reciprocally (e.g., AM/1OCT+).

For both groups, the odor preference ranging from −1 to 1 was calculated. To this end, the number of animals observed on the AM side (#AM) minus the number of animals observed on the 1OCT side (#1OCT) was determined, divided by the total number (#TOTAL)Embedded Image(1)

To determine whether these preferences vary according to the training regimen (i.e., whether they reflect associative memory), the data from alternately run, reciprocally trained groups were taken, and the PI ranging from −1 to 1 was calculated asEmbedded Image(2)

Data for control and experimental groups were gathered alternately.

One-odor learning paradigm in larval Drosophila. In two experimental series, we used a one-odor training regimen (17) by omitting 1OCT from the experiment. That is, the animals in one group received presentations of AM with the reward, alternating with presentations of an empty odor container (EM) on a PURE petri dish (AM+/ EM); the animals trained reciprocally received unpaired presentations of odor and reward (AM/ EM+). During the test, the animals were allowed to choose between AM and EM; the data were then treated, with due adjustments, as described in the preceding section.

Behavior toward odors and sugar in experimentally naïve larval Drosophila. To test for the behavioral specificity of Rhodiola1 treatment, we determined the behavior of experimentally naïve larvae toward the stimuli to be associated for each of the rearing conditions indicated. To test behavior toward FRU, split petri dishes of 85 mm inner diameter were prepared: One-half contained PURE, while in the other half, FRU was present in addition (see sketch in Fig. 2A).

Regarding the odors, the larvae had the choice either between AM and EM or between 1OCT and EM (see sketch in Fig. 2, B and C). In both cases, the larvae were placed in the middle, and after 3 min, the number of larvae on either side was counted; then, the preference index (PREF) values were calculated, with due adjustments, according to Eq. 1.

Olfactory behavior after training-like stimulus exposure in larval Drosophila. As we have argued before (63), the mere exposure to the training stimuli (i.e., odor exposure per se and reward exposure per se) can have nonassociative effects on test behavior. Therefore, the behavior of animals from the control and Rhodiola1 groups toward AM (diluted 1:50 in paraffin oil) and 1OCT, respectively, was assayed after either of two exposure treatments. Either the larvae were exposed to the reward but not to the odors in an otherwise training-like way (see sketches in Fig. 2, D and E) or they were exposed to the odors but not to the reward (Fig. 2, F and G). Then, the PREF scores for AM and 1OCT, respectively, versus EMs were determined, with due adjustments, according to Eq. 1.

Learning experiments in adult Drosophila. Flies were raised and kept on standard fly food (control) or on fly food supplemented with either ground Rhodiola4 root (10 mg/ ml) or FAE-20 (final concentration, 0.71 μM) at 25°C and 60 to 70% relative humidity under a 12-hour light/12-hour dark cycle. For the learning experiments, we used either freshly hatched flies (1 to 3 days after hatching) or 15-day-old flies (after hatching). Every 3 to 5 days after hatching, the “15-day-old” group was transferred to fresh food vials. One day before the behavioral experiments, the flies were starved overnight for 18 to 20 hours at 25°C and 60 to 70% relative humidity in vials equipped with a moist tissue paper to prevent desiccation.

The experimental setup and protocol followed those of (64). As odors, 90 μl of BA (CAS no. 100-52-7, Merck, Darmstadt, Germany) and 340 μl of 3OCT (CAS no. 589-98-0, Merck, Darmstadt, Germany) were applied in 1-cm-deep Teflon containers of 5- and 14-mm diameters, respectively. Two training trials were applied. Each trial started by loading a group of 50 to 100 flies into the setup (0:00 min). One minute later, the flies were transferred to a tube lined with a filter paper that had been soaked the previous day with 2 ml of 2 M SUC solution; then, BA, for example, was shunted into the permanent air flow running through this tube (in half of the cases, 3OCT was used). After 45 s, odor stimulation was terminated, and after an additional 15 s, the flies were taken out of the tube. At the end of a 1-min waiting period, the flies were transferred into another tube lined with a filter paper that had been soaked with pure water the previous day, and the respective other odor was presented. After 45 s, stimulation with this odor was terminated, and 15 s later, the flies were taken out of this second tube. The second trial started immediately. In half of the cases, both training trials started with an odor-sugar presentation; in the other half, both trials started with an odor-alone presentation. After this BA+/ 3OCT training and an additional waiting period of 3 min, the flies were transferred to a T maze, where they could choose between the previously rewarded and the previously unrewarded odor. After 2 min, the choice point of the maze was closed, the flies on each side were counted, and the PREF was calculatedEmbedded Image(3)

A second group was trained reciprocally (BA/ 3OCT+), and the PI was calculated as a measure of associative memory based on their PREF valuesEmbedded Image(4)

The experiments were performed at 22° to 25°C and 75 to 85% relative humidity. Training took place under light; the test was performed in complete darkness, preventing the flies from seeing.

Behavior toward odors and SUC in experimentally naïve adult Drosophila. To test for the behavioral specificity of FAE-20 effects, the preference of control or FAE-20–reared aged flies toward the stimuli to be associated was determined. Flies were tested for responsiveness to the odors BA and 3OCT and to SUC in the same T maze setup used for the learning experiments. To test the preference for olfactory cues, the flies were given 2 min to choose between the two arms of the T maze: one scented with the respective odor used for conditioning and the other one unscented. For each experiment, the number of flies was counted in both arms, and a PREF for each odor was calculated, with due adjustments, according to Eq. 3. To test the preference for SUC, the flies were given 2 min to choose between one arm of the T maze lined with a SUC solution–soaked filter paper and the other arm lined with a water-soaked filter paper. The PREF was calculated, with due adjustments, in the same way.

Statistical analyses of Drosophila behavioral experiments. All statistical analyses were performed with Statistica (version 11; StatSoft Inc., Tulsa, OK, USA). In a conservative approach, nonparametric tests at a statistical significance level of 5% were used throughout. For multiple-group comparisons, Kruskal-Wallis (H) tests were used, followed by pairwise comparisons with Mann-Whitney U tests. For these follow-up pairwise comparisons, the statistical significance level of 5% was maintained with a Bonferroni correction (P < 0.05 divided by the respective number of pairwise tests). Data are displayed as box plots representing the median as the middle line, with the 25th and 75th quantiles as box boundaries and the 10th and 90th quantiles as whiskers.

Quantitative MS of adult Drosophila heads. Single heads of adult Drosophila were resolubilized in 20 μl of water containing 8 M of freshly deionized urea. Tissue and cell destruction was achieved by means of a microglass potter and pulsed sonification on ice for 1 hour. After centrifugation at 21,000g for 15 min at 15°C, 15 μl of the resulting supernatant was transferred to a fresh tube and supplemented with 60 μl of 50 mM NH4HCO3 buffer (pH 8.0) and 2 mM dithiothreitol. After incubation for 1 hour at 20°C, 10 mM methyl methane thiosulfonic acid was added for an additional 1 hour for thiomethylation of previously reduced cysteines. Limited proteolysis was started by adding 250 ng of trypsin (Trypsin Gold, Promega, Mannheim, Germany), followed by incubation at room temperature for 12 hours. Resulting peptides were purified with reversed-phase C18 ZipTip nano-columns (Millipore/Merck, Darmstadt, Germany), eluted with 0.1% trifluoroacetic acid (TFA)/70% acetonitrile (ACN), and dried in a vacuum evaporator centrifuge (Savant, Thermo Fisher Scientific, Waltham, MA, USA).

Proteome analysis was performed on a hybrid dual-pressure linear ion trap/orbitrap mass spectrometer (LTQ Orbitrap Velos Pro, Thermo Fisher Scientific) equipped with an EASY-nLC ultrahigh-performance liquid chromatography (Thermo Fisher Scientific). Samples were resolubilized in 12 μl of 0.1% TFA and 2% ACN and subjected to a 75-μm-inner-diameter, 25-cm PepMap C18 column, packed with 2-μm resin (Thermo Fisher Scientific). Separation was achieved by applying a gradient from 2 to 35% ACN in 0.1% formic acid over a 120-min gradient at a flow rate of 300 nl/min. The LTQ Orbitrap Velos Pro MS used exclusively collision-induced dissociation fragmentation. The spectra acquisition consisted of an orbitrap full MS [Fourier transform MS (FTMS)] scan, followed by up to 15 LTQ tandem MS/MS experiments (TOP15) on the most abundant ions detected in the full MS scan. Essential MS settings were as follows: FTMS (resolution, 60,000; mass/charge ratio range, 400 to 2000) and MS/MS (linear trap; minimum signal threshold, 500; dynamic exclusion time setting, 30 s; singly charged ions were excluded from selection). Normalized collision energy and activation time were set to 35% and 10 ms, respectively.

Raw data processing and protein identification were performed by PEAKS Studio 8.0 (Bioinformatics Solutions; Waterloo, Canada). False discovery rate was set to <1%.

BRP quantification was performed on the basis of the six most abundant tryptic BRP peptides within the MS datasets obtained. Relative protein quantification was achieved using the Skyline analysis platform (65) for MS peak integration on extracted ion chromatograms of the following selected peptide masses:

(1) TQGTLQTVQER: 630.83082+ (precursor), 631.33222+ (precursor [M + 1]), 631.83352+ (precursor [M + 2]), and 632.33472+ (precursor [M + 3]).

(2) SLQTQGGGAAAAGELNK: 786.90252+ (precursor), 787.40392+ (precursor [M + 1]), 787.90522+ (precursor [M + 2]), and 788.40642+ (precursor [M + 3]).

(3) VTYELER: 455.23742+ (precursor) and 455.73892+ (precursor [M + 1]).

(4) LQQSSVSPGDPVR: 685.35712+ (precursor), 685.85862+ (precursor [M + 1]), 686.35992+ (precursor [M + 2]), 686.86112+ (precursor [M + 3]), and 687.36242+ (precursor [M + 4]).

(5) LLQLVQMSQEEQNAK: 879.95642+ (precursor), 880.45782+ (precursor [M + 1]), and 880.95872+ (precursor [M + 2]).

(6) IEMEVQNMESK: 669.30742+ (precursor) and 669.80882+ (precursor [M + 1]).

The monoisotopic precursor mass and one or more 13C-isotopic variants ([M + 1], [M + 2] …) were chosen for more accurate and confident quantification. The peak qualities of the quantified peptides were controlled by the “isotope dot product” (idotp), set to >0.95. Idotp provides a measure for precursor isotope distribution and the correlation between the expected and the observed pattern, with optimal matching resulting in an idotp value of “1” (66).

To analyze the relative abundance of the BRP protein in 2×BRP baseline, 4×BRP control, and 4×BRP flies fed with FAE-20, the six BRP peptides showing the highest intensities of all BRP peptides in the MS raw data were taken into consideration. Having separated by gender each of these six peptide intensities, data from n = 7 to 9 replicates were normalized to the median of the respective peptide intensity of the female or male control. For each group of flies, normalized data of the six peptides were pooled and analyzed with nonparametric statistics.

In all cases, experimenters were blinded to the treatment conditions (food supplementation, genotypes of the animals). These were decoded only after the experiments.

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