Alcohol has a strong causal relationship with sexual arousal and disinhibited sexual behavior in humans; however, the physiological support for this notion is largely lacking and thus a suitable animal model to address this issue is instrumental. We investigated the effect of ethanol on sexual behavior in Drosophila. Wild-type males typically court females but not males; however, upon daily administration of ethanol, they exhibited active intermale courtship, which represents a novel type of behavioral disinhibition. The ethanol-treated males also developed behavioral sensitization, a form of plasticity associated with addiction, since their intermale courtship activity was progressively increased with additional ethanol experience. We identified three components crucial for the ethanol-induced courtship disinhibition: the transcription factor regulating male sex behavior Fruitless, the ABC guanine/tryptophan transporter White and the neuromodulator dopamine. fruitless mutant males normally display conspicuous intermale courtship; however, their courtship activity was not enhanced under ethanol. Likewise, white males showed negligible ethanol-induced intermale courtship, which was not only reinstated but also augmented by transgenic White expression. Moreover, inhibition of dopamine neurotransmission during ethanol exposure dramatically decreased ethanol-induced intermale courtship. Chronic ethanol exposure also affected a male's sexual behavior toward females: it enhanced sexual arousal but reduced sexual performance. These findings provide novel insights into the physiological effects of ethanol on sexual behavior and behavioral plasticity.

The fruit fly Drosophila melanogaster, which offers vast genetic resources, tools and databases, is an excellent model to investigate the physiological mechanisms underlying behavior and has been adopted for studying addictive substances such as alcohol, cocaine, and methamphetamine [10] – [12] . Ethanol is naturally present in fermented fruits and cereals where fruit flies are usually found. Upon exposure to ethanol vapor, flies show increased locomotor activities and sedation [11] . Moreover, flies develop tolerance to the sedative effect of ethanol after a single exposure to high concentrations of ethanol or after a prolonged exposure to low concentrations, which is mediated by adaptive changes in brain activities [13] , [14] . These biphasic and adaptive responses of flies to ethanol are strikingly similar to those of rodents and humans. This implies that ethanol affects the fly and mammalian nervous systems in a similar manner. In this study, we have explored whether recurring ethanol experience elicits behavioral changes in Drosophila. We report here that Drosophila males, upon repeated exposure to ethanol, not only developed tolerance to the sedative effect, but they also displayed active intermale courtship and behavioral sensitization to this effect. Moreover, the neural factor regulating male sexual behavior Fruitless M (Fru M ), the ABC guanine/tryptophan transporter White and the neuromodulator dopamine were crucial in the ethanol-induced courtship disinhibition.

Ethanol acts on multiple neural systems to produce diverse behavioral responses [1] – [3] . At low doses, ethanol induces euphoria and disinhibition whereas excessive consumption causes loss of motor control, sedation and sometimes fatality. A prominent euphoric response associated with ethanol in humans is sexual arousal. The enhanced arousal, in combination with the negative effect of ethanol on cognition, is believed to cause disinhibited sexual behavior, which possibly underlies risky sexual behavior such as unprotected sex and assaults associated with drinking [4] – [6] . The ethanol-associated sexual behavior appears to be due to expectancy (outcome based on learned anticipation) as well as pharmacological effects [5] ; however, physiological evidence is lacking. Animal studies investigating ethanol's effects on sexual behavior have mainly focused on sexual performance, in which ethanol negatively affects copulatory behavior [7] , [8] . Nonetheless, two studies specifically explored ethanol's effect on sexual motivation or arousal in male rats, but their findings are inconsistent [7] , [9] . Therefore, the physiological underpinning of ethanol's effect on sexual arousal and disinhibition needs to be resolved.

Contrary to the effect of chronic ethanol, the initial ethanol experience has a negative effect on male courtship behavior toward females. Without ethanol, males actively courted females, which were typically followed by copulation ( Figure 6B ); however, the courtship activity was drastically diminished under the influence of ethanol on the first exposure ( Figure 6A ). Ethanol also affected sexual performance. In the absence of ethanol, approximately 35% of males copulated with females, whereas only a small percentage of the ethanol-naïve or chronic-ethanol-treated males copulated with females under the influence of ethanol ( Figure 6B ). Thus, both initial and recurring ethanol treatments have a negative effect on copulation.

(A) Courtship. The wings of CS males or females were clipped to distinguish the sex. The ethanol-naïve or ethanol-treated males were housed with an equal number of virgin females and subjected to ethanol exposure in 95% Flypub. Two-factor ANOVA revealed the significant effects of partner, exposure, and interaction in both sets of experiments (intact male with wing-clipped female: partner effect, F 1,27 = 75.8, p<0.0001; exposure effect, F 1,27 = 93.9, p<0.0001; interaction, F 1,27 = 53.2, p<0.0001; n = 7. Wing-clipped male with intact female: partner effect, F 1,27 = 120.8, p<0.0001; exposure effect, F 1,27 = 239.5, p<0.0001; interaction, F 1,27 = 43.2, p<0.0001; n = 7). Double asterisks, significant difference by planned Student t-tests. (B) Copulation. CS males under the influence of ethanol on the 1 st and 6 th exposures displayed significantly reduced copulation with virgin females (ANOVA, F 2,20 = 47.7, p<0.0001; double asterisk, significant difference by Tukey-Kramer; n = 7).

Courtship of Drosophila females is usually passive. Upon daily ethanol treatment, females did not show courtship toward other females (n = 6). Thus, the effect of ethanol on homosexual courtship is specific to males. We next addressed whether the ethanol-induced intermale courtship is attributable to altered sexual orientation of males. If this were the case, chronic-ethanol-treated males would prefer courting males to females. To test this, the age-matched, ethanol-naïve and chronic-ethanol-treated (daily ethanol treatment for 5 days) CS males were subjected to ethanol exposure in the presence of CS virgin females. In this experiment, the wings of either males or females were clipped to distinguish the sex. Typically, males in the absence of ethanol vigorously court virgin females and readily engage in copulation that lasts approximately 20 min [32] . To differentiate sexual behavior affected by ethanol from basal behavior, males and females were acclimated in separate compartments in Flypub and mixed together immediately after ethanol administration. Under the influence of ethanol, the previously ethanol-naïve males displayed a small, but significant, courtship activity toward females and negligible courtship toward males ( Figure 6A ). In contrast, the chronic-ethanol-treated males exhibited dramatically increased courtship toward females as well as males; nonetheless, a significantly larger number of males courted females than males. It was also noticeable that the courting males routinely changed a courtship partner from a male to a female and vice versa. These observations indicate that recurring ethanol experience does not change sexual orientation of males; rather, it enhances sexual arousal and disinhibition.

(A) TH-GAL4/UAS-Shi ts and DDC-GAL4/UAS-Shi ts males, when subjected to daily ethanol exposure at 32°C to block synaptic output of dopamine neurons, displayed drastically reduced intermale courtship compared to the control mw + /UAS-Shi ts males on all exposures (1 st exposure, F 2,21 = 18.24, p<0.0001; 2 nd , F 2,21 = 35.26, p<0.0001; 3 rd , F 2,21 = 27.02, p<0.0001; n = 7–8). Double asterisks, significant difference by Tukey-Kramer tests. (B) TH-GAL4/UAS-Shi ts males, when subjected to daily ethanol exposure at room temperature, showed intermale courtship activities comparable to those of the control TH-GAL4/UAS-GFP males, indicating that Shi ts expressed in dopamine neurons without the dominant negative activity has no effect on the ethanol-induced intermale courtship (Student t-test on all exposures, p>0.5, n = 7).

Ethanol acts on multiple neural systems; however, the altered intermale courtship activities of w and mw + males point to dopamine and serotonin as primary culprits for the ethanol-induced courtship disinhibition. To test this, we employed the GAL4/UAS system [28] and temperature-sensitive dominant negative Dynamin Shi ts [29] to manipulate dopamine neuronal activities. At 30°C or higher restrictive temperature, Shi ts inhibits endocytosis and thus blocks synaptic output. Transgenic TH (tyrosine hydroxylase enhancer)-GAL4 or DDC (dopa decarboxylase enhancer)-GAL4 flies express the transcription factor GAL4 in dopamine or dopamine and serotonin neurons, respectively, to activate expression of the gene (e.g. Shi ts ) downstream of UAS [30] , [31] . Thus, we tested TH-GAL4/UAS-Shi ts and DDC-GAL4/UAS-Shi ts males to recurring ethanol exposure at the restrictive temperature, under which condition synaptic output of dopamine and dopamine/serotonin neurons, respectively, is inhibited. Since all transgenes are tagged with mw + as an in vivo transformation marker, mw + /UAS-Shi ts males were used as a control to match the mw + copy number. When subjected to daily ethanol exposure at 32°C, both TH-GAL4/UAS-Shi ts and DDC-GAL4/UAS-Shi ts males showed significantly reduced intermale courtship activities compared to mw + /UAS-Shi ts males on all exposures examined ( Figure 5A ). When tested at room temperature, on the contrary, TH-GAL4/UAS-Shi ts males displayed the ethanol-induced intermale courtship activities comparable to those of the control males ( Figure 5B ). Thus, Shi ts in the absence of dominant negative activities has no effect on the ethanol-induced intermale courtship. These observations indicate that synaptic output of dopamine neurons is required for the ethanol-induced courtship disinhibition.

Interestingly, the ethanol-induced courtship activities of homozygous f06195 (mw + /mw + ) and heterozygous PHSBJb 3 (hs-mw + /+) males with or without heat shock (incubation at 37°C for 1 h once a day for 3 days prior to ethanol exposure) were higher than that of CS males in all exposures except for the first ( Figure 4 ). On the first exposure, significant levels of intermale courtship were detected in mw + /mw + males and hs-mw + /+ males with heat shock (hs-mw + /+ HS) ( Figure 4A ) while the same heat treatment did not induce intermale courtship in CS and w males (n = 6). The w gene is normally expressed in the eye pigment cells and the brain [27] , in which its expression pattern is unknown. Notably, the eye colors of all transgenic mw + males were lighter than that of CS males and the males expressing mw + in the CS (w+) background displayed enhanced ethanol-induced intermale courtship levels similar to those in the w mutant background (data not shown). Therefore, while deficient White in the eye appears to primarily account for the poor ethanol-induced courtship in w males, over- or mis-expressed White may enhance intermale courtship under ethanol possibly by acting on the cellular pathway(s) underlying courtship disinhibition. Taken together, the ABC guanine/tryptophan transporter White is essential for the ethanol-induced courtship disinhibition and its ectopic or increased expression leads to a high propensity to this behavior.

To test whether the w 1118 male's phenotype could be rescued by reinstating White expression, we employed two independent lines f06195 and PHSBJb 3 , which carry the transgenic mini-white (mw + ) gene in the transformation vectors piggyBAC and P-element, respectively [24] , [25] . The mw + gene represents a w genomic clone including 300 bp 5′ and 630 bp 3′ endogenous regulatory sequence but lacking most of the first intron [26] . In addition, the mw + gene in PHSBJb 3 is under the influence of neighboring heat-shock promoter, which induces over-expression of mw + at 37°C [25] . The heterozygous f06195 and PHSBJb 3 males in the w 1118 mutant background (mw + /+ and hs-mw + /+; Figure 4 ; Movie S2 ) exhibited intermale courtship under the influence of ethanol in an exposure-dependent manner. This indicates that White, the protein absent in the w mutant, is essential for the ethanol-induced courtship disinhibition.

We surveyed pre-existing fly mutants to investigate the cellular mechanism underlying the ethanol-induced courtship disinhibition and found a commonly used strain white 1118 (w 1118 ) displaying negligible intermale courtship upon recurring ethanol administration with 70% (n = 6) or 95% ethanol (n = 32; Figure 4 ; Movie S2 ). A similar result was obtained with the independent allele w 1 (n = 6 for both 70% and 95% ethanol), indicating a strong association of the observed phenotype with the w mutation. The w gene encodes an ABC transporter for guanine and tryptophan that are crucial not only for eye pigmentation but also for dopamine (guanine) or serotonin (guanine and tryptophan) biosynthesis [23] . w males whose eyes are depigmented have a normal capacity to detect light as judged by their strong preference for a lighted to a dark area (n = 8; data not shown); nonetheless, the w male's deficient intermale courtship could be due to their potential visual problem. To explore this possibility, we administered daily ethanol to CS males under infrared light, in which flies can't see. In all exposures up to the 6 th , CS males did not show any intermale courtship (n = 6). Thus, visual input is indispensable for the ethanol-induced intermale courtship and a potential visual anomaly of w males may attribute to their deficient response.

fru 1 and fru 3 males showed vigorous intermale courtship in Flypub without ethanol (No EtOH). Upon daily ethanol treatments in 95% Flypub, their courtship activities did not change with additional ethanol treatment while the courtship levels under ethanol were lower than those without ethanol (ANOVA: fru 1 , F 3,27 = 3.24, p = 0.0396, Tukey-Kramer showed a significant difference between No EtOH and 3 rd EtOH; fru 3 , F 3,27 = 16.94, p<0.0001, No EtOH was significantly different from the others by Tukey-Kramer; n = 7). NS, not significant. CS males tested together with fru males as a control showed negligible intermale courtship in the absence of ethanol and on the 1 st ethanol exposure (marked by diamonds); however, their courtship activities were enhanced under the influence of ethanol on the 2 nd and 3 rd ethanol treatments (F 3,27 = 42.86, p<0.0001; double asterisks, significant difference by Tukey-Kramer).

Genetic alterations in somatic sex development are known to cause intermale courtship in Drosophila [21] . The studies described here, in contrast, reveal recurring ethanol exposure as a post-developmental factor affecting male sexual behavior. We asked whether ethanol affects the brain activity in the manner that the altered brain development causes intermale courtship. If this were the case, ethanol would further enhance the intermale courtship activity of fruitless (fru) males defective in Fru M , a neural sex determination factor. To test this, we employed two fru mutant alleles fru 1 and fru 3 , which have abnormal expression of male-specific Fru M in the central nervous system. fru 1 males have an inversion break point 3.3 Kb upstream of the sexually dimorphic P1 promoter, causing altered Fru M expression: subpopulations of Fru M neurons have either less or undetectable Fru M while numerous non-Fru M neurons display ectopic Fru M expression [22] . On the other hand, fru 3 males with a transposon insertion at the second intron in the fru gene have undetectable Fru M expression [22] . Both fru 1 and fru 3 males exhibit active intermale courtship [22] , which we also observed in Flypub without ethanol ( Figure 3 ). When subjected to daily ethanol exposure, the courtship levels of fru 1 and fru 3 males were decreased on the first exposure, which remained unchanged on subsequent exposures ( Figure 3 ). While the inability for fru males to increase levels of intermale courtship upon recurrent ethanol exposure could be due to the behavioral ceiling effect, the intermale courtship level of fru 3 males is significantly lower than that of CS males on the third exposure (two tailed Student t-test, p<0.001). This suggests that normal physiological function of Fru M or the male-specific neural system established by Fru M is crucial for the ethanol-induced intermale courtship.

A salient effect of ethanol is cognitive impairment [5] , [19] , which may account for the disinhibited courtship of sexually aroused males under the influence of ethanol. Thus, we reasoned that the aged males whose cognitive capacity is reduced [20] might exhibit enhanced ethanol-induced courtship disinhibition. When 2 or 4 wk-old males were subjected to daily ethanol exposure, a significantly higher percentage of males showed intermale courtship on the second ethanol exposure compared to 4 day-old males while the difference was less apparent on subsequent exposures ( Figure 2D ). This implies that certain aging-sensitive activities may be related to the ethanol-induced adaptive changes underlying courtship disinhibition. Taken together, recurring ethanol administration induced conspicuous intermale courtship, which represents disinhibited sexual behavior and entails certain adaptive changes prompted by initial ethanol exposure. The significant increases in this activity in the absence of concurrent increases in tolerance imply distinct mechanisms underlying behavioral sensitization to the disinhibition effect and tolerance to the sedative effect of ethanol.

We assessed whether the ethanol-induced intermale courtship is due to changes in aversive male pheromones, which may become attractive upon repeated ethanol exposure. If this were the case, ethanol-treated males would not court ethanol-naïve males while ethanol-naïve males would actively court ethanol-treated males. To test this, the males treated with daily ethanol for 5 days (chronic-ethanol-treated males) were subjected to ethanol exposure in the presence of the decapitated previously-ethanol-naïve males. In another set of experiments, the ethanol-naïve males were housed with the decapitated chronic-ethanol-treated males and tested for their courtship under the influence of ethanol. The chronic-ethanol-treated males displayed active courtship toward the previously-ethanol-naïve males; however, the courtship activity of the naïve males toward the ethanol-treated males was negligible ( Figure 2C ). Thus, the ethanol-induced intermale courtship is unlikely caused by altered male pheromones.

Notably, the ethanol-induced courtship was dynamic ( Movie S1 ): courtship duration of each pair or chain (courters courted by courtees) ranged from a couple of seconds to minutes and new courtship pairs or chains were continuously formed. These dynamic courtship activities typically lasted for 5 to 10 min before the flies began loosing motor control and became sedated. Moreover, courtship chains with the length ranging from 3 to 5 were frequently noticeable on the third and subsequent ethanol challenges while courtship pairs were dominant on the second exposure. To quantify the ethanol-induced courtship activity, we scored the percentage of males engaged in active courtship during a 30 sec period and used an average of 10 consecutive periods (total 5 min) to represent a percentage of courtship for each group. The percentage of CS males engaged in intermale courtship increased with recurring experience of ethanol, reaching a plateau on the 4 th exposure with 95% ethanol ( Figure 2A ), whereas the males subjected to daily mock treatment did not display any intermale courtship (n = 6). Likewise, no intermale courtship activity was detected when the chronic-ethanol-treated males (daily ethanol for 5 or 6 days) were examined in Flypub in the absence of ethanol (n = 6 each). Therefore, the observed intermale courtship requires physiological actions of ethanol. Similar increases in the courtship activity were observed with 70% ethanol, but with lesser extents on the second and third exposures ( Figure 2B ). These observations together indicate that Drosophila males develop behavioral sensitization to the ethanol's effect on courtship disinhibition.

(A) The percentage of males engaged in intermale courtship progressively increased upon additional ethanol treatments in 95% Flypub. Least squares regression showed the significant effect of exposure (r 2 = 0.68, p<0.0001, n = 10). (B) CS males subjected to daily ethanol exposure in 70% Flypub exhibited the exposure-dependent increase in intermale courtship but at the significantly reduced levels compared to those challenged with 95% ethanol. Two-way ANOVA revealed the significant effects of ethanol concentration, exposure, and interaction (concentration effect, F 1,24 = 54.02, p<0.0001; exposure effect, F 3,24 = 138.2, p<0.0001; interaction, F 3,24 = 6.7, p = 0.0019; n = 4). Post hoc two-tailed Student t-test showed the significant difference of the courtship scores on the 2 nd (p<0.005) or 3 rd exposure (p<0.0005) (marked by double asterisks). (C) The chronic-ethanol-treated males displayed active courtship toward the decapitated previously-ethanol-naïve males under the influence of ethanol (chronic to naïve), whereas the ethanol-naïve males, on the 1 st ethanol exposure, displayed negligible courtship toward the decapitated chronic-ethanol-exposed males (naïve to chronic). Two-tailed Student t-test showed a significant difference (p<0.0001, n = 6, marked by double asterisks). (D) Two or 4 wk-old CS males exhibited the increased levels of intermale courtship compared to 4 d-old males when tested in 95% Flypub. Two-way ANOVA revealed the significant effects of age and exposure, and a marginal interaction (age effect, F 2,36 = 16.6, p<0.0001; exposure effect, F 3,36 = 61.7, p<0.0001; interaction, F 6,36 = 2.58, p = 0.035; n = 4). Tukey-Kramer tests showed that the intermale courtship activities of 2 and 4 wk-old males were significantly different from that of 4 d-old males on the 2 nd exposure (marked by double asterisks).

Upon daily ethanol treatments, CS males showed distinct sexual behavior. Typically, Drosophila males vigorously court females that have attractive pheromones with the courtship ritual comprising a sequential act of following, tapping the female's abdomen, wing vibration (courtship song), licking the female's genitalia, and attempted copulation, which eventually leads to copulation [16] , [17] . Drosophila males, on the contrary, rarely exhibit active courtship toward other mature males [18] , which we also observed in the absence of ethanol or on the first exposure to ethanol ( Figure 2A , Movie S1 ). Occasionally, a male attempted to court another male but quickly moved away. Also, a male courtee strongly rejected a courting male ( Movie S1 ). Under the influence of ethanol on the second and subsequent ethanol treatment, however, CS males actively courted other males in the ritual similar to that shown toward females, which represents disinhibited courtship.

(A) Sedation profile. Flies were exposed to ethanol vapor in 70% Flypub (diamond) or 95% Flypub (triangle). MST in 70% Flypub was higher than that in 95% Flypub on all exposures and recurring treatment in both ethanol concentrations increased MST. Two-way ANOVA revealed the significant effects of ethanol concentration and exposure, and a marginal interaction of two factors (concentration effect, F 1,72 = 383.4, p<0.0001; exposure effect, F 5,72 = 40.5, p<0.0001; interaction, F 5,72 = 2.7, p = 0.03; n = 7). Post hoc Tukey-Kramer tests revealed the significant difference of the 1 st from the other exposures in both ethanol concentrations. All data are reported as mean±standard error of the mean. (B) Ethanol concentrations. CS males were subjected to ethanol treatment for 1, 2 or 6 days (1 st , 2 nd , 6 th ) in 95% Flypub and ethanol contents were measured at 16 or 30 min after the onset of ethanol exposure. CS males without ethanol treatment (No EtOH) were used to measure the basal level. There was no significant difference in the ethanol contents of the males on the 1 st , 2 nd and 6 th exposure at 16 min (ANOVA, F 2,11 = 1.75, p = 0.23, n = 4) or 30 min (F 2,11 = 3.98, p = 0.06, n = 4).

To investigate adaptive behavior associated with recurring exposure to ethanol, we developed a novel apparatus Flypub. Flypub is made of a plastic chamber with a clear ceiling for videotaping behavior and an open bottom for administering ethanol. We exposed fully mature (4 to 5 day-old) wild-type Canton-S (CS) males to intoxicating doses of ethanol once a day for 6 days in Flypub. Prior to ethanol exposure, flies were acclimated to the chamber and then the whole unit was gently placed on a Petri dish containing a cotton pad applied with 70% or 95% ethanol. While slowly exposed to ethanol vapor, flies showed sequential behavioral changes: they became hyperactive (fast walking), lost motor control (infrequent movements and frequent falls during walking), and then were sedated (lying on their back). On the first exposure to ethanol vapor in 95% Flypub, all male flies became sedated within 24 min with a mean sedation time (MST) of approximately 16 min, whereas it took longer with 70% ethanol (MST ∼23 min; Figure 1 ). The flies on the second exposure showed a similar activity profile with a delayed sedation time (∼24 min MST with 95% and ∼35 min with 70%), indicating tolerance development to the sedative effect of ethanol. The tolerance level, as measured by MST, did not change significantly with additional ethanol exposure on consecutive days ( Figure 1 ). It is difficult to compare MST observed in Flypub with that reported in other studies, which employ diverse devices, conditions and parameters. Nonetheless, the mean elution time (MET) of wild-type flies exposed to humidified ethanol vapor (50/45 ethanol/air flow) in the 4 foot-long inebriometer is ∼20 min with 40% and 25% increases in MET on the second exposure at 4 h and 24 h intervals, respectively [13] . When measured in the perforated 50 mL Falcon tube with 50% ethanol vapor, on the other hand, MET (time for immobilizing 50% of flies) is ∼16 min with 100% increase in MET on the second exposure at a 4 h interval [15] . Thus, MST on the first exposure and tolerance levels on the second exposure measured in 95% (16 min; 50%) and 70% (23 min; 52%) Flypub are within the range observed in other studies. Notably, the ethanol concentrations measured at 16 or 30 min after administering ethanol were comparable in the males subjected to ethanol treatment for 1, 2 and 6 days ( Figure 1B ). Therefore, tolerance developed upon repeated ethanol exposure is not due to altered ethanol absorption or metabolism, but likely due to adaptive changes in neural activities.

Discussion

In this report, we have shown that Drosophila males, when subjected to repeated exposure to ethanol, display disinhibited courtship toward other mature males, which is enhanced by additional ethanol challenges. Ethanol affects various aspects of sexual behavior in humans: it is known to impair sexual performance and to enhance sexual arousal or motivation [4], [5]. Moreover, disinhibited sexual behavior is highly associated with alcohol consumption; however, the physiological support for this notion is largely lacking and a suitable animal model to address this issue is instrumental. In a rodent model, a low dose of ethanol reinstates copulatory behavior of the male rats that have been repeatedly trained to suppress their sexual response to unreceptive females [33]. Unfortunately, the study did not distinguish whether the disinhibition effect is on sexual arousal/motivation or sexual performance. Scott et al. have followed up this issue and failed to observe disinhibited sexual motivation [9]. The findings described here provide for the first time unambiguous evidence for disinhibited sexual arousal induced by ethanol and behavioral sensitization to this effect.

Disinhibited homosexual courtship has been previously reported on genetic and transgenic mutants. In particular, homosexual courtship is obvious in transgenic males expressing female TransformerF (a key component of somatic sex determination) or transgenic females expressing FruM (a downstream target of Transformer) in distinct neuronal populations, and fru males with aberrant FruM expression [34]–[38]. These studies indicate that sexual orientation and behavior are controlled by the brain circuitry established by FruM during development. The findings described here, on the other hand, unveil a post-developmental experience of recurring ethanol as a key factor affecting sexual behavior of wild-type males. In the absence of ethanol, fru1 and fru3 males with altered or undetectable FruM expression, respectively, display characteristic intermale courtship, whose levels were slightly (fru1) or significantly (fru3) reduced under the influence of ethanol. The male's courtship toward the female is typically initiated by visual and pheromonal input. The ethanol-induced intermale courtship, on the other hand, appears to depend largely on visual input and less on pheromones since CS males exposed to ethanol under infrared light show negligible intermale courtship as noted above. It is conceivable that reduced levels of intermale courtship observed in fru males under ethanol could be attributable to compromised pheromonal input. Alternatively, certain intermale courtship activities associated with abnormal FruM may be negatively affected by ethanol. It is yet unclear how aberrant FruM expression causes increased intermale courtship in fru1 and fru3 males and enhanced understanding of this process should help resolve this issue. Remarkably, repeated ethanol exposure has no effect on enhancing intermale courtship in fru males. FruM is normally expressed not only during development but also at the adult stage [22]. Thus, disinhibited courtship induced by ethanol may recruit a physiological FruM function or a FruM neural circuit established during development or both. Future studies of additional fru alleles or transheterozygotes with different lesions in the fru gene [39] along with temporally and spatially controlled transgenic manipulation of FruM expression should be instrumental to unravel the mechanism by which FruM mediates the ethanol-associated courtship disinhibition.

Ectopic or increased White expression is previously shown to trigger intermale courtship in the transgenic males carrying mw+ gene under the control of heat shock promoter [25]. Indeed, PHSBJb 3 employed in our study was one of the lines used in the previous study wherein homozygous PHSBJb 3 males in the Df(1)w67c2, yellow genetic background exhibit intermale courtship after heat treatment in a densely populated culture bottle. Under the experimental condition (a low density population) and the genetic background (w1118) used in our study, they did not show a significant level of intermale courtship in the absence of ethanol with or without heat shock. Besides its function in body color pigmentation, Yellow in the brain is crucial for male sex development [40]. This implies that a combined action of yellow mutation and ectopic White in PHSBJb 3 males may attribute to the enhanced intermale courtship observed in the previous study. Similarly in our study, the males with transgenic White expression were more susceptible to the ethanol-induced disinhibited courtship compared to CS males. While White has been extensively used as an eye color marker, several studies indicate the significant roles of White in the central nervous system. Notably, w mutants subjected to submaximal training learn poorly in operant heat-box conditioning, in which flies learn to avoid entering a hot temperature-associated chamber, whereas they learn better in classical olfactory conditioning, in which flies lean to avoid the odor associated with electric shock [41]. w mutants also show a reduced sensitivity to general anesthetics enflurane and halothane [27]. These studies demonstrate the distinct roles of White in various types of behavioral plasticity and anesthesia beyond its function in the eye. While it remains to be resolved whether and how White in the brain is involved in ethanol-induced courtship disinhibition, it is at least conceivable that a major action of over-/mis-expressed White is to inhibit the neural system mediating intermale courtship suppression, potentiating the ethanol-induced courtship disinhibition.

Regarding the cellular mechanism underlying the ethanol-induced courtship disinhibition, the biochemical functions of the White ligands guanine and tryptophan suggest dopamine and serotonin as key components. This notion is supported by the observations that certain polymorphisms in hW, the human homologue of w encoding ABCG1, are linked in males to panic and mood disorders, which are associated with abnormal monoamine functions [42], [43]. Consistently, our results reveal an essential role of dopamine neuronal activities (and presumably released dopamine) in courtship disinhibition induced by ethanol. Dopamine plays crucial roles in mediating the locomotor-activating, rewarding, and behavioral sensitization effects of ethanol in mammals [2], [44]. Indeed, ethanol intake increases dopamine levels in several brain areas and adaptive changes in the dopamine transporter and receptors are associated with alcoholism in humans and rodent models [2], [45]–[47]. Thus, dopamine is a key neuromodulator mediating the pleiotropic effects of ethanol, which is processed by various dopamine receptors in distinct neural systems in mammals, and similar mechanisms may underlie the ethanol-induced behaviors in flies.

Dopamine is also implicated in sexual motivation or arousal in humans, rodents, and flies [12], [48]–[50]. Particularly in flies, transgenic males overexpressing vesicular monoamine transporter in DDC-GAL4 neurons or wild-type males fed with methamphetamine display enhanced courtship toward females [12], [50]. While none of the previous studies in flies and mammals have specifically addressed the dopamine's role in disinhibited sexual behavior, it is conceivable that altered dopamine activities induced by ethanol may be responsible not only for enhancing sexual arousal but also for impairing cognition, causing disinhibited courtship. Indeed, dopamine is involved in numerous cognitive processes including attention, goal-directed behavior, and learning and memory [51], [52]. Five receptor subtypes (D1-5) mediate diverse dopamine functions in mammals. Similarly, Drosophila has D1, D2 and D5 receptors and we have recently identified the distinct functions of the D1 receptor dDA1 for punishment and reward memory formations in olfactory conditioning [53]. Future studies uncovering dopamine receptor subtypes involved in an arousal or cognitive aspect of disinhibited courtship should provide substantial insights on the underlying cellular mechanism.

The deficient ethanol-induced courtship in the males with defective FruM or dopamine neuronal activities suggests that FruM and dopamine systems may be functionally connected to each other for regulating male sexual behavior. One pathway could be for dopamine to modulate the FruM neural circuit. Interestingly, both FruM and dDA1 are highly enriched in the mushroom body neurons projecting to the gamma lobe [37], [38], [54]. It is possible that dopamine, upon binding to dDA1, may play a role in modifying the gamma lobe function established by FruM for courtship disinhibition. Alternatively, FruM may regulate dopamine neuronal activities. This could occur through direct or indirect interactions of FruM and dopamine neurons since both neuronal populations project to many overlapping brain areas [37], [55]. It is noteworthy that a dopamine neuron in each hemisphere, which projects to the anterior commissure and anterior brain areas, is positive for FruM expression (our unpublished observation). Future studies clarifying the functional interaction of FruM and dopamine activities are of great importance to delineate the cellular mechanism underlying the ethanol-induced courtship disinhibition.

The observations described here also reveal the dual effects of ethanol on the heterosexual courtship activity, which is reduced under initial ethanol exposure but enhanced upon chronic treatment. The effect of acute ethanol on sexual arousal or motivation has previously been addressed by two studies in rats. When tested for the operant lever-pressing response to get access to receptive females, the ethanol-injected male rats show increased latencies, implying attenuated sexual motivation [9]. On the other hand, the ethanol-injected male rats show increased frequencies to change platforms prior to encountering receptive females, suggesting enhanced sexual motivation [7]. While our study supports the former, ethanol's effect on sexual arousal/motivation may depend on multiple factors including measurement methods and routes of ethanol administration. Nonetheless, a consistent effect of ethanol on heterosexual behavior observed in our studies of flies and the previous findings in rats [7], [8] and humans [5] is the inhibitory effect on sexual performance. It is tempting to speculate that ethanol may act on similar cellular targets in different species. Comparing the ethanol's effects on heterosexual courtship and copulation, chronic ethanol has opposite effects on sexual arousal and performance of male flies, indicating that their underlying processes may be distinct. It is possible that adaptive changes induced by chronic ethanol exposure are necessary to enhance sexual arousal and may overlap with those underlying behavioral sensitization on disinhibited courtship.

In summary, recurring ethanol administration has diverse effects on sexual behavior of Drosophila males including disinhibited intermale courtship, enhanced sexual arousal toward females and decreased sexual performance. We have identified three cellular components FruM, White and dopamine that are crucial for the ethanol-induced courtship disinhibition. These findings support the notion that alcohol-associated sexual behavior is physiological and provides a baseline to further clarify the underlying cellular mechanisms.