The foraging gene affects alcohol sensitivity, metabolism and memory in Drosophila

Abstract

The genetic basis of alcohol use disorder (AUD) is complex. Understanding how natural genetic variation contributes to alcohol phenotypes can help us identify and understand the genetic basis of AUD. Recently, a single nucleotide polymorphism in the human foraging (for) gene ortholog, Protein Kinase cGMP-Dependent 1 (PRKG1), was found to be associated with stress-induced risk for alcohol abuse. However, the mechanistic role that PRKG1 plays in AUD is not well understood. We use natural variation in the Drosophila for gene to describe how variation of cGMP-dependent protein kinase (PKG) activity modifies ethanol-induced phenotypes. We found that variation in for affects ethanol-induced increases in locomotion and memory of the appetitive properties of ethanol intoxication. Further, these differences may stem from the ability to metabolize ethanol. Together, this data suggests that natural variation in PKG modulates cue reactivity for alcohol, and thus could influence alcohol cravings by differentially modulating metabolic and behavioral sensitivities to alcohol.

Keywords: AUD; Drosophila; Foraging; PRKG1; alcohol; cGMP-dependent protein kinase; locomotion; memory; metabolism.

Figure 1.

for does not affect spontaneous open field behavior. (A) The flyGrAM arena consists of 4 circular arenas each filled with 10 flies of different strains. Flies were tracked while being given humidified air for 5 min, ethanol for 10 min, then humidified air for 5 min. (B) Group activity within each arena was analyzed with the FlyGrAM software while recording the flies during the behavioral paradigm. (C) Ctrax was used to estimate the position and orientation of the 10 flies in each arena per frame. (D) The interactive machine learning for automatic annotation of animal behavior (JAABA) was fed per-frame feature information from Ctrax to reveal information about social interaction of the flies. (E-J) Plots depict mean ± standard error. The spontaneous activity levels of flies given humidified air remain at ~15% and show no significant difference between strains. (G) Velocity of flies given humidified air for 5 min is between 1–3mm/s with no significant difference between strains. (H) Flies cover distances between 30–100cm in five minutes, with no significant differences between strains. (I) Less than 5% of the flies touch per second when given humidified air, with no significant differences between strains. (J) More than 60% of the flies show stopping behavior per second when given humidified air, with no significant differences between strains.

Figure 2.

for affects activity in response to ethanol odor. (A,B) During the period of low-dose ethanol exposure, the activity for for^s and for^s2 decreased, whereas the activity level of for^R stayed the same. (C) for^R traveled significantly longer distances during low dose ethanol exposure compared to for^s and for^s2 flies. (D) for^R showed a significantly higher velocity compared to for^s and for^s2 flies. (E,F) Touching behavior increased for all strains compared to open field behavior whereas stopping behavior significantly decreased to less than 60% of the flies stopping per second. for^R stopped significantly less than for^s and for^s2 flies. Plots depict mean ± standard error n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.

Figure 3.

for does not affect recovery from ethanol odor exposure. (A,B) During presentation of humidified air following exposure to low-dose ethanol, the group activity returned to baseline activity with ~300 s, with no significant differences between strains. (C) The total distance travelled (pathlength) after low-dose ethanol exposure returns to baseline behavior in for^R and for^s2 flies, whereas for^s flies still show an increase pathlength compared to baseline. (D) The velocity after low-dose ethanol exposure returns to baseline behavior in for^R and for^s2 flies, whereas for^s flies still show an increase velocity compared to baseline. (E,F) Touch and Stop behavior recover within minutes of humidified air post ethanol odor. for^s flies demonstrate less stopping than both for^R and for^s2 flies. Since there were no consistent differences between rovers and the two sitter strains in any of the metrics reported, we do not attribute these behavioral differences to variation in for. Plots depict mean ± standard error n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.

Figure 4.

for affects behavioral sensitivity to the pharmacological properties of ethanol. (A) The percent activity in response to a high dose of ethanol increases to ~35% for rovers and ~ 45% for sitters. (B) During high dose ethanol exposure the group activity of for^R is significantly lower than for^s and for^s2 flies. (C) for^s2 flies move a significantly smaller distance than for^R and for^s flies. (D) for^s2 flies are significantly slower than for^R and for^s flies. (E,F) All strains showed increased touching and decreased stopping during high-dose ethanol exposure. (E) for^R showed significantly more touching than for^s2 in the first 5 min of ethanol exposure, and significantly more touching than for^s in the second 5 min of ethanol exposure. (F) for^R showed significantly more stopping than for^s flies throughout high dose ethanol exposure. Plots depict mean ± standard error. n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.

Figure 5.

for affects recovery from the pharmacological properties of ethanol. (A) During exposure to humidified air following an intoxicating dose of ethanol, fly activity decreases more rapidly in for^R than in for^s or for^s2 flies. (B) Significantly fewer for^R flies were active compared to for^s and for^s2 flies. (C) There is significantly reduced (C) pathlength and (D) velocity in for^s2 flies compared to for^R and for^s flies (E) Touching behavior remained higher than baseline levels for all strains, and was statistically reduced in for^s2 compared to for^R flies (F) for^R stopping behavior returned to baseline levels whereas for^s and for^s2 flies continued to show significantly less stopping compared to baseline. Plots depict mean ± standard error. n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.

Figure 6.

for affects memory for ethanol reward. (A) Flies received 3 training sessions with a 10 min exposure to one odor followed by a 10 min exposure to a second odor paired with 60% ethanol vapor. Training trials were spaced by 1 h. A reciprocal group, in which the opposite odor was paired with ethanol, was run. (B) After the training flies were given the choice of the two odors. A preference index was calculated by subtracting the number of flies entering the Odor− vial from the Odor + vial and dividing this number by the total number of flies. Conditioned preference index was calculated by averaging the preference indexes of the two reciprocal groups. (C) for does not affect conditioned aversion tested 30 min after training. (D) for affects conditioned preference tested 24 h after training. for^R flies show decreased memory for ethanol reward compared to for^s and for^s2 flies. (E) for does not significantly affect preference for the odors used in the conditioning procedure. Plots depict mean ± standard error. n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.

Figure 7.

for affects ethanol metabolism. (A) Flies received a 10 min exposure to 60% ethanol in perforated vials in the training boxes used for ethanol memory. Flies from Group One were frozen immediately in liquid nitrogen, flies from group two are exposed to air for another 30 min. Ethanol concentrations in supernatants were measured using an alcohol dehydrogenase–based spectrophotometric assay. To calculate fly internal ethanol concentration, the volume of one fly was estimated to be ?2 ?l. (B) for does not affect ethanol absorption. (C) for affects rate of recovery from ethanol exposure. for^R flies metabolize ethanol faster than for^s and for^s2 flies. Plots depict mean ± standard error. n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p > 0.001, ****=p < 0.0001.