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Keywords:

  • Alcohol;
  • Reward;
  • Aversion;
  • Adolescence;
  • Sex Differences

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

Ethanol (EtOH) abuse is a major health and economic concern, particularly for females who appear to be more sensitive to the rewarding effects of EtOH. This study compared sex differences to the rewarding and aversive effects of EtOH using place-conditioning procedures in rats.

Methods

Separate groups of adult (male, female, ovariectomized [OVX] female) and adolescent (male and female) rats received EtOH (0, 0.5, 1.0, 2.0, or 2.5 g/kg, intraperitoneal) and were confined to their initially nonpreferred side of our conditioning apparatus for 30 minutes. On alternate days, they received saline and were confined to the other side. Following 5 drug pairings, the rats were retested for preference behavior. Separate cohorts of the same groups of rats were injected with a similar dose range of EtOH, and blood EtOH levels (BELs) were compared 30 minutes later.

Results

EtOH produced rewarding or aversive effects in a dose-dependent manner. An intermediate dose of EtOH (1.0 g/kg) produced rewarding effects in adult female, but not in male or OVX female rats, suggesting that ovarian hormones facilitate the rewarding effects of EtOH. Similarly, this intermediate dose of EtOH produced rewarding effects in adolescent female, but not in male rats. The highest dose of EtOH (2.5 g/kg) produced aversive effects that were similar across all adult groups. However, the aversive effects of EtOH were lower in adolescents than adults, suggesting that adolescents are less sensitive to the aversive effects of EtOH. The aversive effects of EtOH did not vary across the estrous cycle in intact adult females. There were also no group differences in BELs, suggesting that our results are not related to EtOH metabolism.

Conclusion

Our results in rats suggest that human females may be more vulnerable to EtOH abuse due to enhanced rewarding effects of this drug that are mediated by the presence of ovarian hormones.

Epidemiological evidence suggests that women are more vulnerable to ethanol (EtOH) abuse. For example, women become intoxicated at faster rates and become EtOH-dependent more readily than men (Greenfield, 2002; Mancinelli et al., 2009). Women are also more likely to develop EtOH-related health problems, such as liver failure, heart attack, cancer, and osteoporosis as compared to men (Epstein et al., 2007). In addition, women display more neurotoxicity and brain damage following EtOH exposure relative to men (Ceylan-Isik et al., 2010; Hommer et al., 1996; Prendergast, 2004). However, women may not be more vulnerable to all aspects of EtOH abuse, as they initiate EtOH use later in life, consume less amounts of EtOH per occasion, and are more likely to remain abstinent after quitting relative to men (York and Welte, 1994). EtOH possesses both appetitive (rewarding) and aversive effects that play a role in EtOH use. The contribution of the rewarding and aversive effects of EtOH to the abuse liability of this drug in women and men has not been well characterized. Thus, this study used a preclinical animal model that compares rewarding and aversive effects of EtOH in female and male rats of different ages.

Preclinical studies in rodents suggest that females experience greater rewarding effects of EtOH as compared to males. For example, females display greater voluntary intake of EtOH versus water in 2-bottle choice procedures in rats (Almeida et al., 1998; Cailhol and Mormede, 2001; Lancaster and Spiegel, 1992; Sluyter et al., 2000; Vetter-O'Hagen et al., 2009) and mice (Middaugh et al., 1999; Tambour et al., 2008). Consistent with this, females display greater EtOH intake than males in operant procedures in rats (Blanchard et al., 1993) and mice (Middaugh and Kelley, 1999). Female rats that were genetically bred to prefer EtOH (P rats) also consume more EtOH than male P rats during peri-adolescence and adulthood under both continuous (Bell et al., 2006) and binge-like (Bell et al., 2011) access conditions. Taken together, these reports suggest that the rewarding effects of EtOH are enhanced in female versus male rodents. However, we also acknowledge reports showing that female mice display similar conditioned place preference (CPP) produced by EtOH as compared to males (Itzhak et al., 2009; Nocjar et al., 1999), and female rats consume less EtOH at higher concentrations as compared to males (van Haaren and Anderson, 1994).

Previous studies have also compared sex differences to the rewarding effects of EtOH in rodents from different stages of development. For example, EtOH-induced CPP was observed in female mice that were tested during the early and late phases of adolescence, whereas males only displayed CPP during the early phase of adolescence (Roger-Sanchez et al., 2012). Adolescent female mice also consume more EtOH compared to their male counterparts (Tambour et al., 2008). In contrast to the latter findings, however, Vetter-O'Hagen and colleagues (2009) showed that adolescent female rats drink less EtOH as compared to adolescent males. In addition, Lancaster and colleagues (1996) found that there were no differences in EtOH drinking in adolescent female and male rats. The inconsistent findings with regard to sex differences in adolescent mice and rats may be related to species-specific differences in the mechanisms that modulate the behavioral effects of EtOH (see Green and Grahame, 2008).

One important aspect to consider when studying adult female rodents is hormone fluctuations that occur across the 4-day estrous cycle (proestrus, estrus, metestrus, and diestrus). A study comparing EtOH self-administration across the estrous cycle observed a reduction in EtOH intake during estrus (Roberts et al., 1998). However, the latter finding was only observed in female rats that were pharmacologically synchronized, but not in females that were allowed to cycle freely. One possible explanation is that estrous cycle synchronization may have elicited behavioral changes, such as arousal and/or hyperactivity that could have interfered with operant behavior given that the synchronized females entered estrus simultaneously. Ford and colleagues (2002a) also showed that EtOH intake was similar across the estrous cycle in freely cycling female rats. Although the behavioral effects of EtOH may not vary across the estrous cycle, we recognize that the neurochemical effects of EtOH may fluctuate across the estrous cycle. This is based on a microdialysis study showing that EtOH produces the largest increase in dopamine levels in the medial prefrontal cortex during estrus when estrogen levels peak (Dazzi et al., 2007).

The influence of hormones on the rewarding effects of EtOH has also been examined using ovariectomy (OVX) procedures. For example, OVX female rats display lower levels of EtOH intake in 2-bottle choice procedures as compared to intact female rats (Almeida et al., 1998; Cailhol and Mormede, 2001). Consistent with this, baseline EtOH intake was significantly reduced following OVX procedures (Ford et al., 2002b). A follow-up study established that the latter effect was reversed with estradiol replacement (Ford et al., 2004). Further validating the importance of ovarian hormones, EtOH-induced increases in dopamine levels in the medial prefrontal cortex are blunted in OVX females as compared to intact females or OVX females that received estrogen pretreatment (Dazzi et al., 2007). These studies highlight the importance of ovarian hormones in mediating the rewarding effects of EtOH in females.

The goal of this study was to examine the influence of sex, age, and ovarian hormones on the rewarding and aversive effects of EtOH. The place-conditioning paradigm assesses the motivational properties of a drug by means of Pavlovian conditioning. EtOH is administered in a distinct environment, and after several pairings, the environmental cues become associated with the effects of the EtOH, thereby acquiring incentive-motivational properties (Tzschentke, 2007). Following conditioning, the environmental cues elicit either approach (CPP) or avoidance (CPA) depending on whether rewarding or aversive effects were elicited by different doses of EtOH during conditioning. The place-conditioning paradigm involves subchronic passive administration of a fixed dose of EtOH, which does not mimic the voluntary intake pattern observed in humans. Procedures involving EtOH drinking assess the appetitive properties of EtOH that are presumed to be evidence of high EtOH reinforcement (Green and Grahame, 2008). Thus, place-conditioning studies assess the association between the rewarding or aversive effects of EtOH with external environmental cues, whereas drinking studies assess the appetitive properties of EtOH. Given our goal of comparing sex differences to the rewarding and aversive effects of EtOH, this study compared place conditioning produced by various doses of EtOH in separate groups of adult male, female, OVX female, and adolescent male and female rats. This study also examined whether the magnitude of aversive effects produced by EtOH varied across the 4-day estrous cycle. Last, blood EtOH levels (BELs) were compared across our experimental groups in order to assess the influence of EtOH metabolism in the results.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Adult (postnatal day [PND] 60 to 75) and adolescent (PND 28 to 45) male and female Wistar rats were handled for 3 days prior to experimentation. The animals were housed in a humidity- and temperature-controlled (22°C) vivarium at the Psychology Department of the University of Texas at El Paso (UTEP). All rats were bred from a stock of outbred Wistar rats from Harlan, Inc. (Indianapolis, IN). Animals were weaned on PND 21 and group housed with same-sex litter mates 2 to 3 per cage. Rats had ad libitum access to standard rodent chow and water, except during conditioning and preference testing. Rats were kept on a reverse light/dark cycle with lights on at 8:00 pm, such that all testing procedures were conducted in the dark phase. All procedures and experimental protocols were approved by the UTEP Institutional Animal Care and Use Committee and were conducted in adherence to the NIH Guidelines for the Care and Use of Laboratory Animals.

Study 1: Place Conditioning

Apparatus

Our conditioning apparatus consisted of 2 rectangular stainless steel chambers (76 × 24 × 30 cm) with 1-way mirrors on the front walls to allow for behavioral observations. Each chamber was divided into 2 distinct compartments of equal proportions that were separated by a removable solid stainless steel partition and elevated above different types of bedding. One compartment had pine bedding beneath a smooth Plexiglas® floor with small holes. The other compartment had green-tinted chlorophyll bedding beneath a metal bar floor. The rats were tested in a dark room under red light with both compartments equally illuminated with white light during the conditioning and testing procedures. It is recognized that the white light may have produced anxiety in the apparatus. However, these conditions were kept constant for all experimental groups, and therefore, do not likely explain any observed group differences. While white light during the dark cycle can induce some phase/circadian-shifting, which may in turn influence daily fluctuations in hormone levels, these possibilities will need to be addressed with multiple control conditions in future studies. Background noise was provided during conditioning and testing by an industrial air filter system that minimized outside disturbances (approximately 50 dB; Honeywell Inc.). Female and male rats were run in separate cohorts to avoid the influence of pheromones on our results. Following conditioning, each apparatus was thoroughly cleaned with Wex-cide detergent, 70% EtOH, and then water.

Experimental Groups

Study 1 compared place conditioning produced by various doses of EtOH (0, 0.5, 1.0, 2.0, or 2.5 g/kg, intraperitoneal [i.p.]) in male (n = 11 to 20 per group) and female (n = 11 to 30 per group) and OVX female (n = 8 to 14 per group) adult rats. Adult rats received the initial preference test on PND 60, conditioning began on PND 67, and the final preference test was conducted on PND 77. In order to compare sex differences in adolescent rats, place conditioning produced by the same doses of EtOH was also compared in adolescent male (n = 6 to 10 per group) and female (n = 7 to 9 per group) rats. Adolescent rats received the initial preference test on PND 28, conditioning began on PND 34, and the final preference test was conducted on PND 45.

Conditioning Procedures

This study employed a biased conditioning procedure consisting of 3 phases: an initial preference test, 5 conditioning trials, and a final preference test. A biased procedure was used because it produces reliable and repeatable results in our laboratory. In our assessment of the literature, more than half of the studies examining EtOH CPP in rats use a biased procedure. Our conditioning procedures are similar to previous reports examining EtOH CPP in rats (for a review, see Fidler et al., 2004).

A pretest was first conducted in order to determine the rats' initially nonpreferred side. Rats were later conditioned with EtOH in their initially nonpreferred side, as defined by the side where they spent <50% of their time during the pretest. However, rats that spent <35% of their time on the nonpreferred side during the pretest were eliminated from the study. This criterion was employed because it is difficult to detect a shift in time spent in a compartment where an animal has a strong initial bias prior to conditioning. This is particularly important for studies employing place-conditioning procedures with drugs that possess weak reinforcing properties such as EtOH in rats.

During preference testing, all rats were placed in the middle of the apparatus. For consistency, all rats were placed into the apparatus facing the same chamber (chlorophyll bedding with metal bar floor). They were then allowed to shuttle freely between the 2 compartments for 15 minutes, a time of testing that has been widely used in our laboratory and that of others (see Fidler et al., 2004; Torres et al., 2008, 2009). A short test period was used to avoid habituation to the test chamber, which facilitates the expression of preference behavior that is based on previous drug environment associations. Rats were considered to have entered a compartment if their 2 front paws were placed on the floor of that compartment. An observer that was blind to the animals' treatment condition scored the time spent in each compartment.

The first conditioning day was conducted 6 days after the initial pretest. The rationale for the delay period was to attempt to minimize the effects latent inhibition on conditioning. Although there is no specific rationale for using a 6-day period, the delay was intended to separate the exposure to the drug-paired compartment in the absence of EtOH during the pretest. This is important given that the purpose of conditioning is to pair the effects of EtOH with the same environmental cues that the rats were exposed to during the pretest. Furthermore, a recent report showed that preexposure to the CPP apparatus before conditioning eliminated CPP produced by EtOH in rats (1.0 g/kg; Morales et al., 2012). Despite this, it is recognized that many laboratories examining EtOH CPP employ more than 1 pretest, as a single baseline trial may be an index of exploratory behavior. To address this issue, we conducted a study comparing the magnitude of CPP produced by EtOH (1.0 g/kg) in female rats (n = 16 to 18 per group) given 1 or 2 pretests. The data revealed that there were no significant differences in the magnitude of CPP in rats that received 1 (128 ± 26) versus 2 (103 ± 16) pretests. These values approximate those depicted in Fig. 1. These data also revealed that the amount of activity was similar on both sides of the conditioning apparatus, suggesting that activity levels do not predict the magnitude of CPP produced by EtOH (data not shown).

image

Figure 1. Place conditioning produced by various doses of ethanol (0, 0.5, 1.0, 2.0, or 2.5 g/kg, i.p.) in adult male (n = 11 to 20), adult female (n = 8 to 30), and OVX female (n = 7 to 14), adolescent male (n = 6 to 10), and adolescent female (n = 7 to 9) rats. The data are presented as difference scores (±SEM), which reflect time spent in the initially nonpreferred side after conditioning minus before conditioning such that values above “0” reflect a positive shift in preference (CPP) and values below “0” represent an a negative shift in preference (CPA). Asterisks (*) denote a significant difference from respective saline controls, daggers (†) reflect a difference from respective male counterparts at a particular dose, and number signs (#) reflect a difference from respective adult counterparts at a particular dose (< 0.05).

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During conditioning, a solid partition separated the chambers so that the rats could be confined to 1 side of the conditioning apparatus. The rats were injected with saline or EtOH and were placed immediately into their initially nonpreferred side for 30 minutes. The saline injections were for the control rats. Separate groups of rats received different doses of EtOH. On alternate days, the rats received saline and were confined to their initially preferred side for 30 minutes. This 2-day procedure was repeated over 10 consecutive days. The order of drug treatment was counterbalanced such that half of the rats from each treatment group received EtOH on the first day of conditioning and the other half received EtOH on the second day of conditioning. Control groups received saline on both days of conditioning. The EtOH solutions were prepared from 95% EtOH diluted in 0.9% sterile saline.

The day after the last conditioning session, a final preference test was conducted. All animals were allowed to shuttle freely between the 3 distinct compartments of our conditioning apparatus for 15 minutes.

Estrous Determination

After the final preference test, adult female rats received vaginal lavage procedures to determine the phase of the estrous cycle they were in during the final preference test (i.e., proestrus, estrus, metestrus, or diestrus). Adolescent females were not subjected to the lavage procedures due to the undifferentiated nature of their epithelium cells. Adolescent females of the age range in the present study are not yet regularly cycling (Forbes and Dahl, 2010; Sisk and Foster, 2004). A sterile and disposable plastic pipette was filled with 0.9% saline and was used to collect epithelial cells. Epithelial cells were then transferred to a labeled glass microscope slide. Microscope slides were fixed with methylene blue stain (Sigma-Aldrich, Inc., St. Louis, MO) and viewed under a light microscope at 40× to examine the shape of the cells and determine the phase of the estrous cycle by the following criteria: proestrus = presence of round nucleated epithelium cells, estrus = presence of cornified un-nucleated epithelium cells, metestrus = presence of leukocytes, and diestrus = limited presence of epithelium cell and leukocytes. A greater number of intact females were included in the 2.5 dose group so that fluctuations across the estrous cycle could be studied at a high EtOH dose. A high dose of EtOH was used to assess behavioral fluctuations across estrous because this dose produced the most robust behavioral effects, and this increased the likelihood of detecting behavioral differences across estrous.

OVX Procedures

The OVX procedure was conducted in young female rats (PND 40 to 45) that were sedated using isoflurane gas. An incision 5 to 8 mm long was made at a point about 1 cm anterior to the knee and 2 cm ventral to the spinal cord. The tissue was separated through the inner layers of connective tissue, and then the ovaries were isolated and ligated. The ovaries were then cut away from the oviduct distal to the ligature. The ends of the oviduct were then placed back inside the body cavity followed by suturing the connective tissue and skin. Animals then received flunixin (2.5 g/kg, subcutaneous) and were allowed to recover for 15 days prior to testing. After the final preference test, an additional 4 days of vaginal lavage procedures verified that our OVX procedure prevented estrous cycling.

Study 2: EtOH Plasma Levels

Study 2 compared BELs in separate groups of naïve rats. Adult (male, intact female, and OVX female) and adolescent (male and female) rats (n = 4 to 10 per group) received a similar dose range of EtOH (0.5, 1.0, or 2.0 g/kg) that was used in Study 1. Thirty minutes later, blood samples were collected from tail veins and centrifuged for 15 minutes at 5,000×g at 4°C. Plasma was then stored at −80°C until analyzed using an Analox AM1 instrument for determining BELs in rodent plasma (Analox Instruments, Lunenburg, MA).

Statistical Analysis

Difference scores were used as the dependent measure, which reflect a shift in preference from the pre- to posttest for each animal. The difference scores were calculated as the amount of time spent in the initially nonpreferred compartment after conditioning minus before conditioning, such that positive values reflect rewarding effects whereas negative values reflect the aversive effects of EtOH. CPP was operationally defined as a significant increase in the difference score obtained from EtOH-treated rats as compared to control rats that received saline during conditioning. In contrast, CPA was defined as a significant decrease in the difference score obtained from EtOH-treated versus control rats.

Our statistical analyses of difference scores (Fig. 1) and BELs (Fig. 3) included overall analyses of variance (ANOVAs) with EtOH dose and group (intact adult female, OVX female, adult male, adolescent male, and adolescent female) as between subject factors. Our statistical analyses of difference scores across the estrous cycle (Fig. 2) included an overall 1-way ANOVA across groups of rats that were tested during different phases of estrous. Where appropriate, significant overall effects were followed by individual post hoc comparisons using Fisher's LSD tests (< 0.05). The comparisons that were made between different groups are denoted with different symbols in the graphs. In Fig. 1, the asterisks (*) denote a significant difference from respective saline controls, daggers (†) reflect a difference from respective male counterparts at a particular dose, and number signs (#) reflect a difference from respective adult counterparts at a particular dose. In Fig. 3, the asterisks (*) denote a significant difference from the lowest dose of EtOH.

image

Figure 2. Place conditioning produced by ethanol (2.5 g/kg) in adult females that were tested during estrus (n = 8), diestrus (n = 5). metestrus (n = 5), or proestrus (n = 6). The data are presented as difference scores (±SEM), which reflect time spent in the initially nonpreferred side after conditioning minus before conditioning such that values below “0” represent a negative shift in preference (CPA).

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image

Figure 3. Plasma blood ethanol (EtOH) levels (BELs) 30 minutes after administration of various doses of EtOH (0.5, 1.0, or 2.0, g/kg, i.p.) in adult male (n = 5 per dose), adult female (n = 4 to 5 per dose), OVX female (n = 4 to 5 per dose), adolescent male (n = 10 per dose), and adolescent female (n = 5 to 6 dose) rats. The asterisks (*) denote a significant increase in BELs relative to the lowest dose of EtOH (0.5 g/kg) collapsed across treatment groups (< 0.05).

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Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Figure 1 displays the effects of various doses of EtOH (0, 0.5, 1.0, 2.0, or 2.5 g/kg, i.p.) in adult (male, intact female, and OVX female) and adolescent (male and female) rats. The results revealed a significant interaction of EtOH dose and group, F(16, 270) = 2.17, < 0.05. Subsequent post hoc analyses revealed that intact adult females conditioned with the 1.0 g/kg dose of EtOH displayed a significant positive shift in preference (CPP) as compared to saline controls (*< 0.05), and the magnitude of this effect in females was significantly higher than adult males and OVX females (†< 0.05). CPP was also observed in adolescent females conditioned with the 1.0 g/kg dose of EtOH versus saline controls (*< 0.05), and the magnitude of this effect was significantly higher in adolescent females versus adolescent males (†< 0.05). A significant negative shift in preference (CPA) was observed in all adult groups that were conditioned with the 2.0 and 2.5 g/kg dose of EtOH as compared to their respective controls (*< 0.05). However, CPA was only observed in adolescent rats that were conditioned with the 2.5 g/kg dose of EtOH versus saline controls (*< 0.05). Furthermore, the degree to which the highest dose of EtOH produced CPA in adolescent male and female rats was lower than their respective adult counterparts (#< 0.05).

One concern is whether OVX procedures influenced our behavioral outcomes. To address this issue, an additional group of female rats (n = 7 to 12) received sham OVX procedures and were conditioned with various doses of EtOH (0, 1.0, and 2.5 g/kg), as described previously (data not shown). The results revealed that there were no differences in the behavioral effects of EtOH across sham and OVX rats, F(1, 87) = 0.04, = 0.84. Namely, similar difference scores were observed in sham OVX (0 = 15.43 ± 19; 1.0 g/kg = 132.4 ± 40; 2.5 g/kg = −118.3 ± 36) and intact (0 = 28.9 ± 23; 1.0 g/kg = 128.16 ± 25; 2.5 g/kg = −158.56 ± 22.4) female rats.

Figure 2 displays avoidance behavior in intact female rats conditioned with the 2.5 g/kg dose of EtOH and tested during different phases of the estrous cycle. The results revealed that there were no differences in avoidance behavior produced by EtOH across the various phases of the estrous cycle, F(3, 20) = 0.96, = 0.42. In order to further examine whether preference behavior was altered across the estrous cycle, an additional analysis was performed that collapsed the data across all doses of EtOH (data not shown). This analysis allowed us to compare behavioral effects with more rats per estrous condition (estrus n = 11; diestrus n = 12; metestrus n = 15; proestrus n = 17). The analysis confirmed that there were no significant differences in preference behavior across the estrous cycle, F(3, 51) = 0.32, > 0.81.

Figure 3 displays BELs following acute administration of various doses of EtOH (0.5, 1.0, or 2.0 g/kg, i.p.) in separate groups of adult (male, intact female, and OVX female) and adolescent (male and female) rats. The results revealed that there was no interaction between EtOH dose and group, F(8, 75) = 0.9, p = 0.5. However, there was a main effect of EtOH dose, F(2, 75) = 90.4, < 0.001, suggesting that EtOH produced an increase in BELs in a dose-dependent manner across all groups. Adult rats that received the 1.0 and 2.0 g/kg dose of EtOH displayed an increase in BELs as compared to rats that received the 0.5 g/kg dose of EtOH (*< 0.001). Adolescent rats that received the 2.0 g/kg dose of EtOH also displayed an increase in BELs as compared to rats that received the 0.5 g/kg dose of EtOH (*< 0.001).

One concern is that the BEL data reflect group differences in EtOH pharmacokinetics following the first exposure to EtOH. However, the rats in Study 1 received 5 injections of EtOH during conditioning. Thus, an additional study compared BELs in response to a single injection of EtOH (1.0 g/kg) in naïve rats versus rats that had previously received 5 injections of the same dose of EtOH (n = 5 to 10 per group). The results revealed that there were no statistically significant differences in BELs in naïve rats (115.8 ± 5.0 mg/dl) versus animals that were preexposed to EtOH (123.6 ± 6.4 mg/dl, = 0.4; data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The main finding of this report is that the rewarding effects of EtOH are enhanced in female rats. To summarize, adult and adolescent female rats displayed enhanced CPP produced by an intermediate dose of EtOH (1.0 g/kg) as compared to males that did not show CPP at any dose of EtOH. The rewarding effects of EtOH appear to be mediated by the presence of ovarian hormones, as OVX females did not display CPP at any dose of EtOH. Our findings are not likely related to sex differences in EtOH metabolism, as there were no differences in BELs across groups of rats that received different EtOH doses. This lack of group differences is consistent with previous reports showing similar BELs across age (Walker and Ehlers, 2009) and sex (Silveri and Spear, 2000) shortly after EtOH administration.

Our findings are consistent with previous reports showing enhanced rewarding effects of EtOH in female versus male rodents. For example, adult females voluntarily consume more EtOH as compared to males in 2-bottle choice procedures in rats (Cailhol and Mormede, 2001; Lancaster and Spiegel, 1992; Lancaster et al., 1996; Sluyter et al., 2000; Vetter-O'Hagen et al., 2009) and mice (Middaugh et al., 1999; Tambour et al., 2008). Adult females also perform more operant responses for EtOH as compared to male rats (Blanchard et al., 1993) and mice (Middaugh and Kelley, 1999). Taken together with the present findings, these studies suggest that the rewarding effects of EtOH are enhanced in female rodents.

Another major finding of this study is that the rewarding effects of EtOH were absent in OVX females. This finding suggests that the presence of ovarian hormones is important for the expression of EtOH reward in adult females. Consistent with this, OVX female rats display lower levels of EtOH consumption relative to their intact counterparts (Almeida et al., 1998; Cailhol and Mormede, 2001; Ford et al., 2002b, 2004). In addition, EtOH produces an increase in dopamine levels in the medial prefrontal cortex of intact female rats, an effect that is blunted in OVX females (Dazzi et al., 2007). The present study also found that the aversive effects of EtOH were similar in intact and OVX female rats. Taken together, our findings suggest that the presence of ovarian hormones influences the rewarding, but not aversive effects of EtOH.

There are several candidate ovarian hormones that may modulate the present findings. A recent review paper supports the importance of estrogen in modulating enhanced vulnerability to several different drugs of abuse in females (Becker et al., 2012). The role of estrogen in mediating the rewarding effects of EtOH in females is supported by the finding that estrogen replacement reestablishes high levels of EtOH intake in OVX female rats (Ford et al., 2004). Estrogen replacement also normalizes EtOH-induced dopamine release in the prefrontal cortex of OVX females (Dazzi et al., 2007). Future studies involving replacement procedures are needed to strengthen our conclusion regarding the role of specific hormones, such as estrogen in facilitating the rewarding effects of EtOH in female rats.

The present study also revealed that the aversive effects produced by a high dose of EtOH were similar in adult females that were tested during different phases of the estrous cycle. This finding suggests that hormonal fluctuations do not influence the aversive effects of EtOH. This is consistent with a report showing that operant responding for EtOH was similar across the estrous cycle in freely cycling female rats (Roberts et al., 1998). However, the possibility exists that place-conditioning and/or operant procedures are not sensitive enough to detect the influence of hormonal fluctuations on the behavioral effects of EtOH. Indeed, Ford and colleagues (2002a) conducted a microstructural analysis of EtOH intake and found that bout frequency was increased during proestrus relative to all other phases of the estrous cycle. Future studies are needed to examine the influence of hormonal fluctuations across the estrous cycle, particularly in rats receiving a dose of EtOH that produces rewarding effects in adult female rats.

The present study also revealed that adolescent females display enhanced rewarding effects of EtOH versus adolescent males. This finding is consistent with the observation that adolescent female mice display CPP produced by EtOH in the early and late phases of adolescence, whereas males only display CPP produced by EtOH in the early phase of adolescence (Roger-Sanchez et al., 2012). Our findings are also consistent with studies showing that adolescent females consume more EtOH compared to adolescent male rats (Truxell et al., 2007) and mice (Tambour et al., 2008). Researchers employing EtOH-drinking procedures interpret high drinking levels to reflect the strong rewarding effects of EtOH. Our data showing that adolescent rats display reduced aversive effects suggest that young animals may also be consuming high levels of EtOH due to reduced sensitivity to the aversive effects of EtOH. Taken together, these studies suggest that adolescent females display increased sensitivity to the rewarding effects of EtOH as compared to adolescent males. Our finding that adolescent females that are not yet regularly cycling display enhanced EtOH CPP suggests that there may be other (nonovarian hormone) mechanisms that contribute to the strong rewarding effects of EtOH in adolescent females. These might include cholinergic and/or amino acid systems that have been shown to promote drug use in adolescent rats (Spear, 2000; Trauth et al., 2000).

The present study also found that EtOH did not produce CPP in male rats (see Figs 1 and 3). Our lack of preference behavior is in line with previous reports in adult male rats (Blatt and Takahashi, 1999; Jones et al., 2009). Although CPP may be difficult to establish with EtOH in naïve rats, previous studies have shown that pretreatment with EtOH before conditioning facilitates this effect in male rats (Bienkowski et al., 1995, 1996; Biala and Kotlinska, 1999; Ciccocioppo et al., 1999; Gauvin and Holloway, 1991; Maldonado-Devincci et al., 2010; Quintanilla and Tampier, 2011) and mice (Nocjar et al., 1999). Regarding the lack of CPP in adolescent males, a previous report demonstrated that adolescent male mice did not show EtOH-induced CPP when they were tested at PND 45, which is the same age that the adolescent rats were tested in our study (Roger-Sanchez et al., 2012). However, the latter study reported EtOH-induced CPP in early adolescent mice that were tested at PND 30. Consistent with this, EtOH-induced CPP has been reported in juvenile rats that were tested at PND 25 (Philpot et al., 2003). Future studies focusing on developmental differences to EtOH-induced CPP might include adolescent male and female rats from an earlier stage of development (less than PND 45).

In conclusion, our data suggest that adult and adolescent females display enhanced sensitivity to the rewarding effects of EtOH relative to males. Our findings also suggest that the presence of ovarian hormones facilitates the rewarding effects of EtOH in females. Thus, heightened vulnerability to EtOH abuse in human females may be the result of enhanced sensitivity to the rewarding effects of EtOH. Future work is needed to better understand the influence of ovarian hormones on the neural mechanisms that mediate enhanced vulnerability to EtOH abuse among females.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors thank Dr. Luis A. Natividad for his helpful comments during the preparation of this manuscript. The authors would also like to thank Arturo Orona, Francisco Roman, Vanessa Valenzuela, and Adrian Muñiz for their technical assistance. This research was supported by the UTEP Office of Research and Sponsored Projects, the Minority Access to Research Careers Program (2T34GM008048), and the Bridges to the Baccalaureate Program (5R25GM049011-12). This research was conducted with the support of faculty and students that are funded by The National Institute on Drug Abuse (R01-DA021274, R24-DA029989, and R25-DA033613) and The National Institute of Minority Health Disparities (G12MD007592). Student funding was also provided by the UTEP Dodson Doctoral Fellowship Program (OVT).

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  6. Acknowledgments
  7. References
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