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Adult anxiety-related behavior of rats following consumption during late adolescence of alcohol alone and in combination with caffeine
Alcohol 45 (2011) 365e372
Adult anxiety-related behavior of rats following consumption during late adolescence of alcohol alone and in combination with caffeine Robert N. Hughes* Department of Psychology, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Received 1 April 2010; received in revised form 20 September 2010; accepted 12 October 2010
Abstract During late adolescence (postnatal days, PNDs, 45e55), male and female hooded rats were exposed to alcohol (1.14e1.33 g/kg/day), caffeine (27.03e27.22 mg/kg/day) or alcohol and caffeine together (1.20e1.34 g/kg/day alcohol plus 23.85e26.48 mg/kg/day caffeine) via their drinking water. The rats’ anxiety-related behavior was then assessed on reaching mid adulthood at PND120 in a lightedark box and an open field. For males only, alcohol alone led to increased entries of the lightedark box and (compared with water- or caffeine-exposed subjects) open-field rearing. Alcohol and caffeine combined also increased entries of the lightedark box light compartment and openfield ambulation for males only. The drug combination led to more male ambulation than for alcohol alone, and higher occupancy of the center squares of the apparatus than for males in any other group. Although alcohol alone had no subsequent effects on female behavior, alcohol and caffeine combined led to fewer entries of and less time spent in the lightedark box side then females in any other group. The drug combination also led to less female ambulation in the open field compared with either water- or caffeine-exposed females. The results were interpreted as sex-related potentiation by caffeine of alcohol’s developmental effects that resulted in lower levels of adult anxiety in male, but higher levels in females. The possible significance of this outcome for humans, especially females, was discussed. Ó 2011 Elsevier Inc. All rights reserved. Keywords: Alcohol; Caffeine; Rats; Adolescence; Anxiety; Sex differences
Introduction It is now widely acknowledged that the human adolescent brain is not fully mature but is in a stage of transition from childhood to adulthood (Blakemore and Choudhury, 2006; Casey et al., 2008; Dahl, 2004; Smith, 2003) particularly with regard to prefrontal and limbic structures that involve the action of dopamine, glutamate, gammaaminobutyric acid, and serotonin (Spear, 2000). Consequently, during adolescence the brain is far more vulnerable than during adulthood to long-term changes induced by drugs particularly because of overproduction followed by reduction (synaptic pruning) of monoaminergic synapses and receptors (Anderson and Navalta, 2004). This greater vulnerability is enhanced by adolescents’ higher levels of sensation-seeking and risky behavior (Arnett, 1992), which can predispose them to experiment with mind-altering substances to a greater extent than during adulthood. Although some adolescents may begin this experimentation with illicit drugs, the majority will initially focus on legal * Corresponding author. Tel.: þ64-3-364-2879; fax: þ64-3-364-2181. E-mail address: [email protected] (R.N. Hughes) 0741-8329/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2010.10.006
substances, namely alcohol and nicotine (Kandel and Yamaguchi, 1993). Regular alcohol consumption by adolescents is very widely practiced (Bonomo et al., 2001; Lewinsohn et al., 1996; Li et al., 1996; Spear, 2002; Sutherland and Willner, 2002) with problem drinking during adolescence being associated with problem drinking in adulthood (McCarty et al., 2004). There is good evidence from animal research that exposure during adolescence to alcohol orally, by injection or as an inhaled vapor, can affect subsequent brain/behavior development. Long-lasting alteration of brain functioning has been observed (Crews et al., 2000; Ehlers and Criado, 2010; Maldonado-Devincci et al., 2010; Nagel et al., 2005; Witt, 2010) with accompanying behavioral changes. These changes have been in the form of increased (Slawecki et al., 2004) or decreased anxiety (Salimov et al., 1996) and impairments of spatial learning and memory (Sircar and Sircar, 2005) and other indices of cognitive performance (Pascual et al., 2007). Such observations are consistent with reports of persisting neurocognitive dysfunction in humans following alcohol abuse during adolescence (Guerri and Pascual, 2010). Mechanisms proposed for the impairments observed in animals have
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included increased cell death in the neocortex, hippocampus, and cerebellum (Pascual et al., 2007); changes in activity of frontal cortical NMDA receptors (Sircar and Sircar, 2006); and inhibition of neurogenesis (Crews et al., 2006). However, in rodent research, the focus has largely been on alcohol-induced increases in brain dopamine with particular reference to the nucleus accumbens and possible long-term effects on neuronal functioning (Maldonado-Devincci et al., 2010). Many teenagers also consume caffeine on a daily basis (Hughes and Hale, 1998) particularly in energy or soft drinks. Alcohol and caffeine are often taken together by adding a caffeinated mixer (such as Red Bull, Coca Cola, or Relentless) to an alcoholic drink. In fact, during the last decade, there has been an increasing preference for caffeinated alcoholic drinks among young adults (Marczinski and Fillmore, 2006) many of whom would still be undergoing the final stages of adolescent brain development (Spear, 2000). As there have been reports of the action of alcohol being either antagonized or potentiated by caffeine (Fudin and Nicastro, 1988; Holloway and Holloway, 1979; White, 1999), it is important to know what might be the consequences for subsequent behavioral development of mixing the two drugs together. Although there is a reasonable literature on the consequences for cognitive development of adolescent exposure to alcohol (Brown and Tapert, 2004; Smith, 2003), less attention has been paid to emotional development. However, both decreased and increased anxiety has been implicated in the developmental effects of adolescent exposure to alcohol in rats (Salimov et al., 1996; Slawecki et al., 2004). Therefore, the present study was designed to assess the later effects on anxiety-related behavior of treating both male and female rats with alcohol alone or combined with caffeine at a stage of development approximately equivalent to when most humans begin drinking alcoholecaffeine mixtures, namely late adolescence. Apart from one study in which pregnant rats fed liquid diets containing both drugs produced fewer and lighter offspring (Hannigan, 1995), there appears to have been little attention paid to the developmental consequences of exposure to alcoholecaffeine combinations. The inclusion of female rats in the present investigation was especially important in view of reports of greater vulnerability to a number of effects of alcohol shown by human females than by males (Baraona et al., 2001; Holdcroft and Iacono, 2002; Mann et al., 2005). In addition, female adolescents are increasing their alcohol consumption and are also beginning to drink at an earlier age than previously (Spear, 2002). This is of concern because there are sex differences in adolescent brain maturation (De Bellis et al., 2001) and, as suggested by the bloodeoxygen-dependent response to a spatial working memory task, it is also possible that the female adolescent brain is accordingly more vulnerable to alcohol-induced deficits (Caldwell et al., 2005). It was also important to study female rats because of the dominance of male-only
rodent investigations of effects of alcohol and other drugs (Hughes, 2007; Zucker and Beery, 2010). To more closely approximate human consumption of alcohol and caffeine and avoid any stressful more invasive form of administration, both drugs alone and in combination were provided in the rats’ communal drinking water during late adolescence, namely from 45 to 55 days after birth. The rats were caged in same-sexed groups of three or four individuals to avoid possible confounding effects of social isolation (Hatch et al., 1963). The choice of doses of alcohol and caffeine aimed for (1.0e1.5 g/kg and 20e30 mg/kg, respectively) was guided by previous research in which developmental consequences had followed treatment during adolescence with both drugs within these dose ranges (e.g., Anderson and Hughes, 2008; Sircar and Sircar, 2006). Materials and methods Subjects The subjects were 40 male and 40 female Piebald Virol Glaxo/C (PVG/C) rats from 20 litters of comparable sizes. When they were 28 days old, they were weaned and housed in cages of three or four same-sexed rats. Each rat in a cage was from a different litter. They were allowed free access to food and water in a room with an ambient temperature of 22 6 2 C and a 12 h light/dark cycle (lights on at 0800 h). When the rats reached late adolescence (PND45), the water in each water bottle was replaced with the appropriate solutions of alcohol, caffeine, or alcohol plus caffeine for each cage, except for those assigned to the control condition. Two cages containing three and one cage containing four same-sexed rats were randomly assigned to each of the three drug exposure conditions. Although there are differing opinions about the age boundaries of adolescence in rats (Spear, 2000), ages of around PNDs 40e55 are generally accepted as encompassing late adolescence (Laviola et al., 2004; Mathews and McCormick, 2007; Walker et al., 2009) when biological and behavioral changes characterizing the transition from adolescence to adulthood (such as attaining reproductive maturity, increased social interactions, greater novelty seeking) may still be in progress (Adriani et al., 1998; Primus and Kellogg, 1989; Smith, 2003). Procedures for housing, exposure to alcohol and caffeine, and testing of all subjects complied with Parts 5 (Codes of Welfare) and 6 (Use of Animals in Research, Testing, and Teaching) of the New Zealand Animal Welfare Act (1999), and had been approved by the Animal Ethics Committee of the University of Canterbury. Experimental drinking fluids On the basis of average volumes of water drunk by each cage from PND40 to PND42, estimates were made of the quantities of alcohol and caffeine that needed to be added to the drinking water to give average daily doses per rat
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of 1.0e1.5 g/kg alcohol and 20e30 mg/kg caffeine alone or combined together. These quantities were calculated to be 8.5 g/L drinking fluid for alcohol and 170 mg/L for caffeine. Every second day from PND45 (when treatment began) until PND55, all rats were weighed and an average weight for their cage was calculated. The volume of fluid drunk from the communal drinking bottle in each cage was also measured to enable subsequent determination of the average daily doses/rat of alcohol, caffeine, and alcohol plus caffeine that each cage of rats was exposed to during late adolescence (PNDs45e55). Although these averages proved useful in relating consumption to subsequent behavioral outcomes, it was not possible to guarantee that each individual rat in a cage drank the same volume of solution in proportion to bodyweight because of possible competition for the drinking tube from each communal water bottle. However, earlier research has suggested that such competition is minimal and that two or more rats have been seen drinking simultaneously or in quick succession from the same tube (Wolffgramm, 1990). Behavioral testing When the rats reached PND120 (mid adulthood), half the rats experienced a lightedark box test followed by a test in an open field. The other half experienced the reverse sequence. Each set of apparatus was cleaned with a 2% solution of Powerquat Blue disinfectant between the end of testing for one rat, and the beginning for the next. Lightedark box testing Preferences for the dark versus light half of a lightedark box were assessed in apparatus consisting of two 300-mm long 200-mm wide 300-mm high compartments separated from each other by a wooden partition. A rat was able to move between the two sides through a 100-mm 100-mm opening in the center of the partition that could be closed by means of a removable horizontal slide. The dark side was covered by a hinged wooden lid, whereas the light side was covered by a clear Perspex lid. Each rat was placed in the dark side for approximately 30 s with the slide separating the two sides inserted. Then, the slide was withdrawn and the rat was allowed free access to both sides. For exactly 5 min, the total time spent in and the number of entries of the light side was recorded. Lower levels of time spent in and fewer entries of the light side are commonly viewed as indicative of higher anxiety (Belzung, 1999; Hasco€et et al., 2001).
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Open-field testing The open-field tests were conducted in a 600 600-mm Perspex open field with clear 300 mm-high walls, and a black floor divided into 16 numbered squares by a grid of white intersecting lines. The field sat on a 700-mm high table beneath dim fluorescent room lighting (47l). Each rat was placed in the center of the apparatus and, every 3 s for exactly 5 min, it was noted which square it was occupying, and whether or not it was rearing on its hind legs. On completion of the trial, fecal boluses left in the apparatus were counted and removed. From the square occupancy data, it was possible to subsequently estimate the distance traveled (ambulation) by counting the number of times an occupied square differed from that occupied during an immediately preceding 3-s observation (Hughes and Beveridge, 1987). It was also possible to count the number of times a rat was seen to be occupying one of the four center squares of the apparatus. Higher rates of defecation and lower rates of rearing, ambulation, and center square occupancy are often viewed as indicative of higher anxiety in rats (Archer, 1973; Belzung, 1999; Brain and Marrow, 1999; Lister, 1991). Data analysis The average volumes of fluid drunk by rats in each cage were subjected to a 4 (drug exposure) 2 (sex) analysis of variance (ANOVA). Sex differences in the average doses of each drug that were consumed alone or in combination were assessed by separate t tests. All behavioral measures in each of the two types of apparatus were analyzed by separate 4 (drug exposure) 2 (sex) ANOVAs. Whenever significant exposure effects occurred, post hoc comparisons between groups were made with Fischer Protected Least Significant Difference (PLSD) tests (P ! .05). Results Fluid consumption and drug doses Average fluid consumption/cage for each exposure condition and sex was calculated in proportion to 100 g average bodyweight/cage, and is outlined in Table 1. Although differences between the exposure conditions were not significant (F[3,16] 5 0.87, P O .1), the average volume/cage of fluid drunk by female rats was significantly greater than that drunk by males (F[1,16] 5 24.96, P ! .0001). From the average volumes/cage of solution drunk, average doses/cage of alcohol and caffeine
Table 1 Mean 6 standard error of the mean values of the average fluid/100 g average bodyweight drunk during adolescence for cages of rats that were provided with unadulterated communal drinking water or water containing alcohol, caffeine, or alcohol þ caffeine, and by males and females Drug exposure
Fluid consumption
Sex
Control
Alcohol
Caffeine
Alcohol þ caffeine
Males
Females
9.67 6 0.88
10.89 6 1.21
11.23 6 0.99
10.54 6 1.03
8.82 6 0.50
12.36 6 0.46
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Table 2 Mean 6 standard error of the mean average doses/per average bodyweight consumed during adolescence of alcohol (alone and combined with caffeine) and caffeine (alone and combined with alcohol) for males and female separately Exposure condition
Males
Females
Alcohol alone (g/kg) Alcohol with caffeine (g/kg) Caffeine alone (mg/kg) Caffeine with alcohol (mg/kg)
1.14 6 0.02 1.20 6 0.02 27.03 6 0.78 23.85 6 1.18
1.33 6 0.01 1.34 6 0.02 27.22 6 1.38 26.48 6 0.44
consumed/day alone and in combination were determined for each sex, and are shown in Table 2. However, it must be emphasized that volumes of solution drunk and thus exact doses of each drug consumed could not be determined for individual rats and are thus estimates based on averages for cages of rats. Females consumed significantly more alcohol than males when provided either alone (t[4] 5 7.98, P ! .005) or when combined with caffeine (t[4] 5 4.80, P ! .01). There were no significant sex differences for caffeine consumed alone (t[4] 5 0.12, P O .9) or when combined with alcohol (t[4] 5 2.10, P O .1). Lightedark box results Although the adolescent exposure main effect was not significant for either entries of (F[3,72] 5 1.76, P O .1) or time spent in the light side (F[3,72] 5 1.56, P O .2), the exposure sex interaction was significant in both cases (entries, F[3,72] 5 3.37, P ! .025; time, F[3,72] 5 2.77, P ! .05). As shown in Fig. 1A, male rats exposed to alcohol alone or when combined with caffeine entered the light side significantly more often than water-exposed males, whereas female rats exposed to the drug mixture entered this side significantly less often than females exposed to either water or alcohol alone. Females exposed to alcohol and caffeine combined also spent significantly less time in the light side than those in any of the other three conditions, but there were no significant exposure effects on this measure for males (see Fig. 1B). For all rats combined, females entered the light side significantly more often (F[1,72] 5 17.92, P ! .0001) and spent more time in it than males (F[1,72] 5 4.88, P ! .035). Open-field results The exposure main effect was not significant for either open-field ambulation (F[1,72] 5 0.27, P O .8) or rearing (F[3,72] 5 0.02, P O .9), but there were significant exposure sex interactions for both measures (ambulation, F[3,72] 5 3.06, P ! .035; rearing, F[3,72] 5 2.79, P ! .05). These revealed significantly more ambulation for males following exposure to the alcohol and caffeine mixture than exposure to either water or alcohol alone (see Fig. 2A).
Fig. 1. Mean 6 standard error of the mean entries of (A), and time spent in (B) the light side of the lightedark box for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. abGroups with superscripts in common significantly different (P ! .05) for that particular sex.
However, females exposed to the mixture displayed significantly less ambulation than those exposed to either water or caffeine alone. Rearing occurred significantly more often for males (but not females) exposed to alcohol alone, whereas females exposed to caffeine alone reared significantly less often than those exposed to water (see Fig. 2B). For all rats combined, females displayed significantly more ambulatory and rearing activity than males (ambulation, F[1,72] 5 16.42, P ! .0001; rearing, F[1,72] 5 24.70, P ! .0001). The main exposure effect for occupancy of the center squares of the apparatus was not significant (F[3,72] 5 2.76, P O .06), but there was again a significant exposure sex interaction (F[3,72] 5 2.76, P ! .05), which was accounted for by significantly greater occupancy for male
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Fig. 2. Mean 6 standard error of the mean open-field ambulation (A), and rearing (B) for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. a Groups with superscript in common significantly different (P ! .05) for that particular sex.
rats exposed to the drug mixture than to either water or alcohol alone (see Fig. 3A). Females were not affected by their adolescent experience. The number of fecal boluses left in the apparatus was not significantly affected by adolescent exposure to the experimental drinking fluids (F[3,72] 5 0.25, P O .8, see Fig. 3B) but, for all rats combined, females left significantly fewer boluses than males (F[1,72] 5 47.88, P ! .0001). Discussion The higher average fluid consumption/100 g bodyweight shown by female rats no doubt arose from their tendency to drink more fluid than males because of their lower levels of circulating hormones that are associated with antidiuresis
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Fig. 3. Mean 6 standard error of the mean occupancy of open-field center squares (A), and numbers of fecal boluses left in the apparatus (B) for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. ab Groups with superscripts in common significantly different (P ! .05) for that particular sex.
(McGivern et al., 1996). Although the doses consumed of alcohol alone and when combined with caffeine were higher for females than for males, it was extremely unlikely that the differences (0.19 and 0.14 g/kg) were large enough to be pharmacologically significant. Similar sex differences in rats’ alcohol consumption have been reported by other authors (Juarez and de Tomasi, 1999) and these have been related to higher levels of estrogen or greater release of nucleus accumbens dopamine in females than in males (Blanchard et al., 1993; Lancaster et al., 1996). Although it was clear that treatment with alcohol alone or combined with caffeine during late adolescence had effects on subsequent adult behavior, the nature of most of these effects was dependent on the sex of the rats. As
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reflected in entries of the light side of the lightedark box and rearing in the open field, alcohol on its own appeared to decrease anxiety for males (but not females) compared with water or caffeine. A similar effect on anxiety following adolescent treatment with alcohol was reported by Salimov et al. (1996) for alcohol-preferring male rats, although reduced inhibition has also been proposed as a reason for more approaches of a novel object (Stansfield and Kirstein, 2007). However, the opposite outcome for anxiety-related behavior was later observed with nonpreferring rats (sex and strain unspecified but probably male SpragueeDawley rat, Slawecki et al., 2004). When alcohol was combined with caffeine, both sexes were affected by their adolescent experience, but in different ways. By entering the light compartment of the lightedark box more often, engaging in more open-field ambulation and occupying the center squares of the apparatus more often, male rats exposed to the drug mixture appeared to display less anxiety-related behavior than those exposed to water or, in the case of open-field ambulation and center squares occupancy, alcohol. For the latter measure, males exposed to the combination also appeared less anxious than those exposed to caffeine alone. Females on the other hand, appeared to be more anxious following exposure to the drug mixture as reflected in fewer entries of and less time spent in the light compartment of the lightedark box compared with females exposed to either water or alcohol alone. Further evidence of increased anxiety for females following exposure to the mixture was found in their lower level of open-field ambulation than those exposed to water or caffeine. When alcohol alone decreased one index of anxiety in males (i.e., entries of the lightedark box light side), the addition of caffeine did not change this apparently alcohol-related anxiolytic effect. But when alcohol had no effect on other anxiety indices for this sex (i.e., open-field ambulation and center squares occupancy), the addition of caffeine appeared to decrease anxiety compared with exposure to alcohol alone. However for females, a rather different pattern of results emerged. Although adolescent exposure to alcohol alone did not produce any subsequent effects in adulthood, the addition of caffeine led to the opposite effect to what occurred with males. As indicated by decreased entries of and time spent in the lightedark box light side, females exposed to the alcoholecaffeine combination appeared to have become more anxious than females from any other group. This was also reflected in their lower ambulation scores compared with females exposed to either water or caffeine. In general, for both sexes, rats exposed to caffeine alone did not differ significantly from those exposed to water, except for female rearing. As the caffeine results did not mimic those obtained with alcohol and caffeine combined, the combination’s effects cannot be merely ascribed to the action of caffeine alone. Therefore, the outcomes for both male and female rats demonstrated modification by caffeine of the
developmental effects of alcohol (which may or may not have had sex-related anxiolytic effects in its own right). It would therefore appear that caffeine may have potentiated the effects of alcohol for both sexes but in different directions. This possibility is of considerable interest given that most research in this area has involved male animals only. The decrease in open-field rearing for female rats exposed to caffeine alone resembled a similar decrease for both sexes combined following daily intraperitoneal injections of 15 mg/kg caffeine during adolescence that was ascribed to increased anxiety (Anderson and Hughes, 2008). The lack of the same outcome for male rats in the present study may have been due to differences in how caffeine was administered, namely orally via a communal drinking bottle versus repeated intraperitoneal injections. Although determination of reasons for the sex-related outcomes described in the present paper clearly remain the goal of further research, they demonstrate the need to include both sexes in investigations of this sort especially because increased anxiety in females following adolescent exposure to alcohol and caffeine combined may be a further possible indication of human females’ greater vulnerability to effects of alcohol (Baraona et al., 2001; Holdcroft and Iacono, 2002; Mann et al., 2005). The overall sex differences described whereby, compared with male rats, females entered the lightedark box light compartment more often, and displayed more open-field ambulation and rearing while leaving fewer fecal boluses in the apparatus were consistent with earlier observations of them being more active (Archer, 1975) and less anxious than males (Gray, 1971). Although the measurement of blood alcohol and caffeine levels in adolescent rats given voluntary access to either of the two drugs has not been a general feature of earlier studies (e.g., Anderson and Hughes, 2008; Salimov et al., 1996), future investigations should include such measurements. Because of possible inequalities in access to the communal drinking bottle when rats are treated in groups, this would provide an indication of the extent to which individual animals consequently varied in their drug intake. Although there is good reason to expect greater vulnerability of adolescent rats to the consequences of exposure to drugs compared with older animals (Smith, 2003; Spear, 2002), it is nevertheless necessary to address the possibility that the results of the present study may not have been specific to this particular developmental period. Therefore, because much higher doses of alcohol than those used in the present study can differentially induce brain damage in adolescent and adult rats (Crews et al., 2000), it is nevertheless still important to also investigate subsequent effects of exposure to lower doses of alcohol and caffeine both before and after adolescence. Although there is obviously a need for much more research, the results of the present study nevertheless suggest that the consumption of mixed alcoholic and caffeinated beverages by adolescent females, might lead to more undesirable consequences in adulthood, than consumption of alcohol on its own.
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Acknowledgment Assistance of Amber Rowse in the drug treatment of rats and the collection of data is gratefully acknowledged.
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Adult anxiety-related behavior of rats following consumption during late adolescence of alcohol alone and in combination with caffeine Robert N. Hughes* Department of Psychology, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Received 1 April 2010; received in revised form 20 September 2010; accepted 12 October 2010
Abstract During late adolescence (postnatal days, PNDs, 45e55), male and female hooded rats were exposed to alcohol (1.14e1.33 g/kg/day), caffeine (27.03e27.22 mg/kg/day) or alcohol and caffeine together (1.20e1.34 g/kg/day alcohol plus 23.85e26.48 mg/kg/day caffeine) via their drinking water. The rats’ anxiety-related behavior was then assessed on reaching mid adulthood at PND120 in a lightedark box and an open field. For males only, alcohol alone led to increased entries of the lightedark box and (compared with water- or caffeine-exposed subjects) open-field rearing. Alcohol and caffeine combined also increased entries of the lightedark box light compartment and openfield ambulation for males only. The drug combination led to more male ambulation than for alcohol alone, and higher occupancy of the center squares of the apparatus than for males in any other group. Although alcohol alone had no subsequent effects on female behavior, alcohol and caffeine combined led to fewer entries of and less time spent in the lightedark box side then females in any other group. The drug combination also led to less female ambulation in the open field compared with either water- or caffeine-exposed females. The results were interpreted as sex-related potentiation by caffeine of alcohol’s developmental effects that resulted in lower levels of adult anxiety in male, but higher levels in females. The possible significance of this outcome for humans, especially females, was discussed. Ó 2011 Elsevier Inc. All rights reserved. Keywords: Alcohol; Caffeine; Rats; Adolescence; Anxiety; Sex differences
Introduction It is now widely acknowledged that the human adolescent brain is not fully mature but is in a stage of transition from childhood to adulthood (Blakemore and Choudhury, 2006; Casey et al., 2008; Dahl, 2004; Smith, 2003) particularly with regard to prefrontal and limbic structures that involve the action of dopamine, glutamate, gammaaminobutyric acid, and serotonin (Spear, 2000). Consequently, during adolescence the brain is far more vulnerable than during adulthood to long-term changes induced by drugs particularly because of overproduction followed by reduction (synaptic pruning) of monoaminergic synapses and receptors (Anderson and Navalta, 2004). This greater vulnerability is enhanced by adolescents’ higher levels of sensation-seeking and risky behavior (Arnett, 1992), which can predispose them to experiment with mind-altering substances to a greater extent than during adulthood. Although some adolescents may begin this experimentation with illicit drugs, the majority will initially focus on legal * Corresponding author. Tel.: þ64-3-364-2879; fax: þ64-3-364-2181. E-mail address: [email protected] (R.N. Hughes) 0741-8329/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2010.10.006
substances, namely alcohol and nicotine (Kandel and Yamaguchi, 1993). Regular alcohol consumption by adolescents is very widely practiced (Bonomo et al., 2001; Lewinsohn et al., 1996; Li et al., 1996; Spear, 2002; Sutherland and Willner, 2002) with problem drinking during adolescence being associated with problem drinking in adulthood (McCarty et al., 2004). There is good evidence from animal research that exposure during adolescence to alcohol orally, by injection or as an inhaled vapor, can affect subsequent brain/behavior development. Long-lasting alteration of brain functioning has been observed (Crews et al., 2000; Ehlers and Criado, 2010; Maldonado-Devincci et al., 2010; Nagel et al., 2005; Witt, 2010) with accompanying behavioral changes. These changes have been in the form of increased (Slawecki et al., 2004) or decreased anxiety (Salimov et al., 1996) and impairments of spatial learning and memory (Sircar and Sircar, 2005) and other indices of cognitive performance (Pascual et al., 2007). Such observations are consistent with reports of persisting neurocognitive dysfunction in humans following alcohol abuse during adolescence (Guerri and Pascual, 2010). Mechanisms proposed for the impairments observed in animals have
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included increased cell death in the neocortex, hippocampus, and cerebellum (Pascual et al., 2007); changes in activity of frontal cortical NMDA receptors (Sircar and Sircar, 2006); and inhibition of neurogenesis (Crews et al., 2006). However, in rodent research, the focus has largely been on alcohol-induced increases in brain dopamine with particular reference to the nucleus accumbens and possible long-term effects on neuronal functioning (Maldonado-Devincci et al., 2010). Many teenagers also consume caffeine on a daily basis (Hughes and Hale, 1998) particularly in energy or soft drinks. Alcohol and caffeine are often taken together by adding a caffeinated mixer (such as Red Bull, Coca Cola, or Relentless) to an alcoholic drink. In fact, during the last decade, there has been an increasing preference for caffeinated alcoholic drinks among young adults (Marczinski and Fillmore, 2006) many of whom would still be undergoing the final stages of adolescent brain development (Spear, 2000). As there have been reports of the action of alcohol being either antagonized or potentiated by caffeine (Fudin and Nicastro, 1988; Holloway and Holloway, 1979; White, 1999), it is important to know what might be the consequences for subsequent behavioral development of mixing the two drugs together. Although there is a reasonable literature on the consequences for cognitive development of adolescent exposure to alcohol (Brown and Tapert, 2004; Smith, 2003), less attention has been paid to emotional development. However, both decreased and increased anxiety has been implicated in the developmental effects of adolescent exposure to alcohol in rats (Salimov et al., 1996; Slawecki et al., 2004). Therefore, the present study was designed to assess the later effects on anxiety-related behavior of treating both male and female rats with alcohol alone or combined with caffeine at a stage of development approximately equivalent to when most humans begin drinking alcoholecaffeine mixtures, namely late adolescence. Apart from one study in which pregnant rats fed liquid diets containing both drugs produced fewer and lighter offspring (Hannigan, 1995), there appears to have been little attention paid to the developmental consequences of exposure to alcoholecaffeine combinations. The inclusion of female rats in the present investigation was especially important in view of reports of greater vulnerability to a number of effects of alcohol shown by human females than by males (Baraona et al., 2001; Holdcroft and Iacono, 2002; Mann et al., 2005). In addition, female adolescents are increasing their alcohol consumption and are also beginning to drink at an earlier age than previously (Spear, 2002). This is of concern because there are sex differences in adolescent brain maturation (De Bellis et al., 2001) and, as suggested by the bloodeoxygen-dependent response to a spatial working memory task, it is also possible that the female adolescent brain is accordingly more vulnerable to alcohol-induced deficits (Caldwell et al., 2005). It was also important to study female rats because of the dominance of male-only
rodent investigations of effects of alcohol and other drugs (Hughes, 2007; Zucker and Beery, 2010). To more closely approximate human consumption of alcohol and caffeine and avoid any stressful more invasive form of administration, both drugs alone and in combination were provided in the rats’ communal drinking water during late adolescence, namely from 45 to 55 days after birth. The rats were caged in same-sexed groups of three or four individuals to avoid possible confounding effects of social isolation (Hatch et al., 1963). The choice of doses of alcohol and caffeine aimed for (1.0e1.5 g/kg and 20e30 mg/kg, respectively) was guided by previous research in which developmental consequences had followed treatment during adolescence with both drugs within these dose ranges (e.g., Anderson and Hughes, 2008; Sircar and Sircar, 2006). Materials and methods Subjects The subjects were 40 male and 40 female Piebald Virol Glaxo/C (PVG/C) rats from 20 litters of comparable sizes. When they were 28 days old, they were weaned and housed in cages of three or four same-sexed rats. Each rat in a cage was from a different litter. They were allowed free access to food and water in a room with an ambient temperature of 22 6 2 C and a 12 h light/dark cycle (lights on at 0800 h). When the rats reached late adolescence (PND45), the water in each water bottle was replaced with the appropriate solutions of alcohol, caffeine, or alcohol plus caffeine for each cage, except for those assigned to the control condition. Two cages containing three and one cage containing four same-sexed rats were randomly assigned to each of the three drug exposure conditions. Although there are differing opinions about the age boundaries of adolescence in rats (Spear, 2000), ages of around PNDs 40e55 are generally accepted as encompassing late adolescence (Laviola et al., 2004; Mathews and McCormick, 2007; Walker et al., 2009) when biological and behavioral changes characterizing the transition from adolescence to adulthood (such as attaining reproductive maturity, increased social interactions, greater novelty seeking) may still be in progress (Adriani et al., 1998; Primus and Kellogg, 1989; Smith, 2003). Procedures for housing, exposure to alcohol and caffeine, and testing of all subjects complied with Parts 5 (Codes of Welfare) and 6 (Use of Animals in Research, Testing, and Teaching) of the New Zealand Animal Welfare Act (1999), and had been approved by the Animal Ethics Committee of the University of Canterbury. Experimental drinking fluids On the basis of average volumes of water drunk by each cage from PND40 to PND42, estimates were made of the quantities of alcohol and caffeine that needed to be added to the drinking water to give average daily doses per rat
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of 1.0e1.5 g/kg alcohol and 20e30 mg/kg caffeine alone or combined together. These quantities were calculated to be 8.5 g/L drinking fluid for alcohol and 170 mg/L for caffeine. Every second day from PND45 (when treatment began) until PND55, all rats were weighed and an average weight for their cage was calculated. The volume of fluid drunk from the communal drinking bottle in each cage was also measured to enable subsequent determination of the average daily doses/rat of alcohol, caffeine, and alcohol plus caffeine that each cage of rats was exposed to during late adolescence (PNDs45e55). Although these averages proved useful in relating consumption to subsequent behavioral outcomes, it was not possible to guarantee that each individual rat in a cage drank the same volume of solution in proportion to bodyweight because of possible competition for the drinking tube from each communal water bottle. However, earlier research has suggested that such competition is minimal and that two or more rats have been seen drinking simultaneously or in quick succession from the same tube (Wolffgramm, 1990). Behavioral testing When the rats reached PND120 (mid adulthood), half the rats experienced a lightedark box test followed by a test in an open field. The other half experienced the reverse sequence. Each set of apparatus was cleaned with a 2% solution of Powerquat Blue disinfectant between the end of testing for one rat, and the beginning for the next. Lightedark box testing Preferences for the dark versus light half of a lightedark box were assessed in apparatus consisting of two 300-mm long 200-mm wide 300-mm high compartments separated from each other by a wooden partition. A rat was able to move between the two sides through a 100-mm 100-mm opening in the center of the partition that could be closed by means of a removable horizontal slide. The dark side was covered by a hinged wooden lid, whereas the light side was covered by a clear Perspex lid. Each rat was placed in the dark side for approximately 30 s with the slide separating the two sides inserted. Then, the slide was withdrawn and the rat was allowed free access to both sides. For exactly 5 min, the total time spent in and the number of entries of the light side was recorded. Lower levels of time spent in and fewer entries of the light side are commonly viewed as indicative of higher anxiety (Belzung, 1999; Hasco€et et al., 2001).
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Open-field testing The open-field tests were conducted in a 600 600-mm Perspex open field with clear 300 mm-high walls, and a black floor divided into 16 numbered squares by a grid of white intersecting lines. The field sat on a 700-mm high table beneath dim fluorescent room lighting (47l). Each rat was placed in the center of the apparatus and, every 3 s for exactly 5 min, it was noted which square it was occupying, and whether or not it was rearing on its hind legs. On completion of the trial, fecal boluses left in the apparatus were counted and removed. From the square occupancy data, it was possible to subsequently estimate the distance traveled (ambulation) by counting the number of times an occupied square differed from that occupied during an immediately preceding 3-s observation (Hughes and Beveridge, 1987). It was also possible to count the number of times a rat was seen to be occupying one of the four center squares of the apparatus. Higher rates of defecation and lower rates of rearing, ambulation, and center square occupancy are often viewed as indicative of higher anxiety in rats (Archer, 1973; Belzung, 1999; Brain and Marrow, 1999; Lister, 1991). Data analysis The average volumes of fluid drunk by rats in each cage were subjected to a 4 (drug exposure) 2 (sex) analysis of variance (ANOVA). Sex differences in the average doses of each drug that were consumed alone or in combination were assessed by separate t tests. All behavioral measures in each of the two types of apparatus were analyzed by separate 4 (drug exposure) 2 (sex) ANOVAs. Whenever significant exposure effects occurred, post hoc comparisons between groups were made with Fischer Protected Least Significant Difference (PLSD) tests (P ! .05). Results Fluid consumption and drug doses Average fluid consumption/cage for each exposure condition and sex was calculated in proportion to 100 g average bodyweight/cage, and is outlined in Table 1. Although differences between the exposure conditions were not significant (F[3,16] 5 0.87, P O .1), the average volume/cage of fluid drunk by female rats was significantly greater than that drunk by males (F[1,16] 5 24.96, P ! .0001). From the average volumes/cage of solution drunk, average doses/cage of alcohol and caffeine
Table 1 Mean 6 standard error of the mean values of the average fluid/100 g average bodyweight drunk during adolescence for cages of rats that were provided with unadulterated communal drinking water or water containing alcohol, caffeine, or alcohol þ caffeine, and by males and females Drug exposure
Fluid consumption
Sex
Control
Alcohol
Caffeine
Alcohol þ caffeine
Males
Females
9.67 6 0.88
10.89 6 1.21
11.23 6 0.99
10.54 6 1.03
8.82 6 0.50
12.36 6 0.46
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Table 2 Mean 6 standard error of the mean average doses/per average bodyweight consumed during adolescence of alcohol (alone and combined with caffeine) and caffeine (alone and combined with alcohol) for males and female separately Exposure condition
Males
Females
Alcohol alone (g/kg) Alcohol with caffeine (g/kg) Caffeine alone (mg/kg) Caffeine with alcohol (mg/kg)
1.14 6 0.02 1.20 6 0.02 27.03 6 0.78 23.85 6 1.18
1.33 6 0.01 1.34 6 0.02 27.22 6 1.38 26.48 6 0.44
consumed/day alone and in combination were determined for each sex, and are shown in Table 2. However, it must be emphasized that volumes of solution drunk and thus exact doses of each drug consumed could not be determined for individual rats and are thus estimates based on averages for cages of rats. Females consumed significantly more alcohol than males when provided either alone (t[4] 5 7.98, P ! .005) or when combined with caffeine (t[4] 5 4.80, P ! .01). There were no significant sex differences for caffeine consumed alone (t[4] 5 0.12, P O .9) or when combined with alcohol (t[4] 5 2.10, P O .1). Lightedark box results Although the adolescent exposure main effect was not significant for either entries of (F[3,72] 5 1.76, P O .1) or time spent in the light side (F[3,72] 5 1.56, P O .2), the exposure sex interaction was significant in both cases (entries, F[3,72] 5 3.37, P ! .025; time, F[3,72] 5 2.77, P ! .05). As shown in Fig. 1A, male rats exposed to alcohol alone or when combined with caffeine entered the light side significantly more often than water-exposed males, whereas female rats exposed to the drug mixture entered this side significantly less often than females exposed to either water or alcohol alone. Females exposed to alcohol and caffeine combined also spent significantly less time in the light side than those in any of the other three conditions, but there were no significant exposure effects on this measure for males (see Fig. 1B). For all rats combined, females entered the light side significantly more often (F[1,72] 5 17.92, P ! .0001) and spent more time in it than males (F[1,72] 5 4.88, P ! .035). Open-field results The exposure main effect was not significant for either open-field ambulation (F[1,72] 5 0.27, P O .8) or rearing (F[3,72] 5 0.02, P O .9), but there were significant exposure sex interactions for both measures (ambulation, F[3,72] 5 3.06, P ! .035; rearing, F[3,72] 5 2.79, P ! .05). These revealed significantly more ambulation for males following exposure to the alcohol and caffeine mixture than exposure to either water or alcohol alone (see Fig. 2A).
Fig. 1. Mean 6 standard error of the mean entries of (A), and time spent in (B) the light side of the lightedark box for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. abGroups with superscripts in common significantly different (P ! .05) for that particular sex.
However, females exposed to the mixture displayed significantly less ambulation than those exposed to either water or caffeine alone. Rearing occurred significantly more often for males (but not females) exposed to alcohol alone, whereas females exposed to caffeine alone reared significantly less often than those exposed to water (see Fig. 2B). For all rats combined, females displayed significantly more ambulatory and rearing activity than males (ambulation, F[1,72] 5 16.42, P ! .0001; rearing, F[1,72] 5 24.70, P ! .0001). The main exposure effect for occupancy of the center squares of the apparatus was not significant (F[3,72] 5 2.76, P O .06), but there was again a significant exposure sex interaction (F[3,72] 5 2.76, P ! .05), which was accounted for by significantly greater occupancy for male
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Fig. 2. Mean 6 standard error of the mean open-field ambulation (A), and rearing (B) for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. a Groups with superscript in common significantly different (P ! .05) for that particular sex.
rats exposed to the drug mixture than to either water or alcohol alone (see Fig. 3A). Females were not affected by their adolescent experience. The number of fecal boluses left in the apparatus was not significantly affected by adolescent exposure to the experimental drinking fluids (F[3,72] 5 0.25, P O .8, see Fig. 3B) but, for all rats combined, females left significantly fewer boluses than males (F[1,72] 5 47.88, P ! .0001). Discussion The higher average fluid consumption/100 g bodyweight shown by female rats no doubt arose from their tendency to drink more fluid than males because of their lower levels of circulating hormones that are associated with antidiuresis
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Fig. 3. Mean 6 standard error of the mean occupancy of open-field center squares (A), and numbers of fecal boluses left in the apparatus (B) for males and females separately following exposure during adolescence to unadulterated drinking water (Water) or water containing caffeine (Caff), alcohol (Alc), or alcohol plus caffeine (Alc þ caff). *Significantly different (P ! .05) from water control group for that particular sex. ab Groups with superscripts in common significantly different (P ! .05) for that particular sex.
(McGivern et al., 1996). Although the doses consumed of alcohol alone and when combined with caffeine were higher for females than for males, it was extremely unlikely that the differences (0.19 and 0.14 g/kg) were large enough to be pharmacologically significant. Similar sex differences in rats’ alcohol consumption have been reported by other authors (Juarez and de Tomasi, 1999) and these have been related to higher levels of estrogen or greater release of nucleus accumbens dopamine in females than in males (Blanchard et al., 1993; Lancaster et al., 1996). Although it was clear that treatment with alcohol alone or combined with caffeine during late adolescence had effects on subsequent adult behavior, the nature of most of these effects was dependent on the sex of the rats. As
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reflected in entries of the light side of the lightedark box and rearing in the open field, alcohol on its own appeared to decrease anxiety for males (but not females) compared with water or caffeine. A similar effect on anxiety following adolescent treatment with alcohol was reported by Salimov et al. (1996) for alcohol-preferring male rats, although reduced inhibition has also been proposed as a reason for more approaches of a novel object (Stansfield and Kirstein, 2007). However, the opposite outcome for anxiety-related behavior was later observed with nonpreferring rats (sex and strain unspecified but probably male SpragueeDawley rat, Slawecki et al., 2004). When alcohol was combined with caffeine, both sexes were affected by their adolescent experience, but in different ways. By entering the light compartment of the lightedark box more often, engaging in more open-field ambulation and occupying the center squares of the apparatus more often, male rats exposed to the drug mixture appeared to display less anxiety-related behavior than those exposed to water or, in the case of open-field ambulation and center squares occupancy, alcohol. For the latter measure, males exposed to the combination also appeared less anxious than those exposed to caffeine alone. Females on the other hand, appeared to be more anxious following exposure to the drug mixture as reflected in fewer entries of and less time spent in the light compartment of the lightedark box compared with females exposed to either water or alcohol alone. Further evidence of increased anxiety for females following exposure to the mixture was found in their lower level of open-field ambulation than those exposed to water or caffeine. When alcohol alone decreased one index of anxiety in males (i.e., entries of the lightedark box light side), the addition of caffeine did not change this apparently alcohol-related anxiolytic effect. But when alcohol had no effect on other anxiety indices for this sex (i.e., open-field ambulation and center squares occupancy), the addition of caffeine appeared to decrease anxiety compared with exposure to alcohol alone. However for females, a rather different pattern of results emerged. Although adolescent exposure to alcohol alone did not produce any subsequent effects in adulthood, the addition of caffeine led to the opposite effect to what occurred with males. As indicated by decreased entries of and time spent in the lightedark box light side, females exposed to the alcoholecaffeine combination appeared to have become more anxious than females from any other group. This was also reflected in their lower ambulation scores compared with females exposed to either water or caffeine. In general, for both sexes, rats exposed to caffeine alone did not differ significantly from those exposed to water, except for female rearing. As the caffeine results did not mimic those obtained with alcohol and caffeine combined, the combination’s effects cannot be merely ascribed to the action of caffeine alone. Therefore, the outcomes for both male and female rats demonstrated modification by caffeine of the
developmental effects of alcohol (which may or may not have had sex-related anxiolytic effects in its own right). It would therefore appear that caffeine may have potentiated the effects of alcohol for both sexes but in different directions. This possibility is of considerable interest given that most research in this area has involved male animals only. The decrease in open-field rearing for female rats exposed to caffeine alone resembled a similar decrease for both sexes combined following daily intraperitoneal injections of 15 mg/kg caffeine during adolescence that was ascribed to increased anxiety (Anderson and Hughes, 2008). The lack of the same outcome for male rats in the present study may have been due to differences in how caffeine was administered, namely orally via a communal drinking bottle versus repeated intraperitoneal injections. Although determination of reasons for the sex-related outcomes described in the present paper clearly remain the goal of further research, they demonstrate the need to include both sexes in investigations of this sort especially because increased anxiety in females following adolescent exposure to alcohol and caffeine combined may be a further possible indication of human females’ greater vulnerability to effects of alcohol (Baraona et al., 2001; Holdcroft and Iacono, 2002; Mann et al., 2005). The overall sex differences described whereby, compared with male rats, females entered the lightedark box light compartment more often, and displayed more open-field ambulation and rearing while leaving fewer fecal boluses in the apparatus were consistent with earlier observations of them being more active (Archer, 1975) and less anxious than males (Gray, 1971). Although the measurement of blood alcohol and caffeine levels in adolescent rats given voluntary access to either of the two drugs has not been a general feature of earlier studies (e.g., Anderson and Hughes, 2008; Salimov et al., 1996), future investigations should include such measurements. Because of possible inequalities in access to the communal drinking bottle when rats are treated in groups, this would provide an indication of the extent to which individual animals consequently varied in their drug intake. Although there is good reason to expect greater vulnerability of adolescent rats to the consequences of exposure to drugs compared with older animals (Smith, 2003; Spear, 2002), it is nevertheless necessary to address the possibility that the results of the present study may not have been specific to this particular developmental period. Therefore, because much higher doses of alcohol than those used in the present study can differentially induce brain damage in adolescent and adult rats (Crews et al., 2000), it is nevertheless still important to also investigate subsequent effects of exposure to lower doses of alcohol and caffeine both before and after adolescence. Although there is obviously a need for much more research, the results of the present study nevertheless suggest that the consumption of mixed alcoholic and caffeinated beverages by adolescent females, might lead to more undesirable consequences in adulthood, than consumption of alcohol on its own.
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Acknowledgment Assistance of Amber Rowse in the drug treatment of rats and the collection of data is gratefully acknowledged.
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