Beber combinados a base de refrescos energéticos con un alto contenido en cafeína y alcohol puede afectar el cerebro de los adolescentes igual que si consumieran cocaína. Además, este hábito durante la juventud puede alterar la capacidad de controlar el consumo de sustancias gratificantes, como el alcohol y las drogas, en la adultez. Investigadores de la Universidad Purdue han constatado estos efectos en ratones adolescentes.
«Parece que ambas sustancias juntas provocan cambios en el comportamiento y en la neuroquímica del cerebro»
Over the last decade, numerous products containing high levels of caffeine have emerged [1,2]. These products include energy drinks, powdered caffeine, caffeine pills, buccal caffeine pouches, caffeinated peanut butter, and caffeine vaporizer sticks. These highly caffeinated products are disproportionally targeted to adolescents and young adults . Of these products, the most widely used are highly caffeinated energy drinks, which come in a variety of different volumes (from 1.7 oz energy shots to 20 oz. cans) and caffeine concentrations (9–170 mg/oz.) [2,4,5]. Sales of energy drinks grew 60% from 2008 to 2013, illustrating the increased popularity and consumption of these beverages. Yet, increased accessibility of highly caffeinated products has coincided with increased reports of emergency departments visits because of energy drink consumption , highlighting the potential harms of exposure to highly caffeinated solutions to adolescents.
While the consumption of large quantities of caffeine itself is problematic [2,7], added health risks arise when caffeine is consumed with alcohol. It has been reported that 23% to 47% of adolescents and young adult alcohol users consume alcohol-mixed energy drinks [8,9]. Surveys of college-aged students suggest this population consumes large amounts of caffeine-mixed alcohol to fulfill hedonistic motives, such as increased pleasure from intoxication and increasing the intensity and/or nature of intoxication [10,11]. However, serious–and sometimes fatal–consequences can occur when mixing caffeine with alcohol [12–14]. While it is clear that consumption of caffeine-mixed alcohol solutions by adolescents and young adults carries a significant acute health risk, the long-term consequences of repeated exposures to caffeine-mixed alcohol are not yet well understood.
The lack of information on the potential long-term risks is particularly concerning given that adolescents, who are the predominant consumers of caffeine-mixed alcohol, are known to be more susceptible to changes in behavioral and neuronal adaptations from exposure to psychostimulants and drugs of abuse than adults [15–17]. Increased responses to cocaine-induced locomotor stimulation and reward have been observed in adolescent mice exposed to caffeine but not in animals exposed to caffeine in adulthood , suggesting chronic exposure outcomes in adolescence are not synonymous with exposures outcomes in adulthood. Legal and ethical issues surrounding alcohol use in minors heavily limits caffeine-mixed alcohol studies in human to self-reported survey-based results or in-laboratory performance tasks [18,19]; yet, animal studies provide a viable option for studying the effects of caffeine-mixed alcohol on adolescent behavior in a controlled setting . Importantly, results observed in previous animal studies correlate with reported effects in adolescents and young adults [17,20–22]. Here we developed an animal model using adolescent mice to mimic exposure to caffeine-mixed alcohol as reported by college-aged adults [6,10,11].
Both caffeine and alcohol are known to increase dopamine release in dopaminergic reward pathways, specifically through their actions involving adenosine and dopamine receptors in the dorsal striatum and nucleus accumbens [23,24]. We hypothesized that repeated consumption of caffeine-mixed alcohol causes stronger activation of the dopaminergic reward pathway than caffeine or alcohol alone and could be on par with the levels of dopamine released by commonly abused psychostimulants, such as cocaine, leading to unique behavioral and pharmacological adaptations. To evaluate how chronic adolescent exposure to caffeine-mixed alcohol alters drug-related behaviors, we exposed C57BL/6 mice to caffeine-mixed alcohol throughout adolescence and monitored changes in locomotor sensitivity, ΔFosB accumulation, cocaine preference, cocaine sensitivity, and natural reward to saccharin. We observed unique behavioral and neurochemical effects of repeated caffeine-mixed alcohol exposure in adolescent mice that may indicate that these animals will experience future events involving caffeine-mixed alcohol, natural rewards, or cocaine and/or other psychostimulants differently than animals not exposed to caffeine-mixed alcohol in adolescence.
Adolescent (approximately postnatal day 28 [P28]) male and female C57BL/6 mice were obtained from Harlan Inc. (Indianapolis IN, USA) and allowed to acclimate for one week to handling and drug administration before behavioral testing began at postnatal day 35 [25,26]. Unless specified otherwise, mice were grouped housed in single grommet ventilated Plexiglas cages at ambient temperature (21°C) in a room maintained on a reversed 12L:12D cycle (lights off at 10.00, lights on at 22.00) in animal facilities, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Food and water were provided ad libitum and mice were not deprived of food or water at any time. All animal procedures were pre-approved by Institutional Animal Care and Use Committees of Purdue University and the University of California San Francisco and conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Caffeine, ethyl alcohol (200 proof), cocaine hydrochloride, and saccharin were obtained from Sigma Aldrich (St. Louis MO, USA). Caffeine (15 mg/kg), alcohol (1.5 g/kg), and caffeine (15 mg/kg) mixed alcohol (1.5 g/kg) solutions were administered via intraperitoneal injection (i.p., diluted in 0.9% saline) or oral gavage (o.g., dissolved in reverse osmosis water). Cocaine (1.5–30 mg/kg, diluted in 0.9% saline) was administered intraperitoneally (i.p.). For transcardial perfusion, a ketamine (Henry Schein Animal Health, Dublin OH, USA) and xylazine (Sigma Aldrich) cocktail of 100:10 mg/kg solution was administered (10 mg/mL i.p.) to induce anesthesia. Phosphate-buffered saline (PBS), 16% paraformaldehyde ampules (Electron Microscopy Sciences, Hatfield PA, USA), and heparin (10 units/mL) (Sigma) were utilized during perfusion. Saccharin solutions were prepared in reverse osmosis water to concentrations of 0.25 mM, 0.5 mM, 1.0 mM, and 2.0 mM.
Adolescent male and female C57BL/6 mice (n = 9–11 per group) were administered saline (0.9%), caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by intraperitoneal injection for either five days a week for two weeks (male only animals, Fig 1A) or four weeks (male and female animals, Fig 1B). Locomotor activity was measured for 60 minutes in locomotor activity boxes (L 27.3 cm x W 27.3 cm x H 20.3 cm, Med Associates, St Albans City VT, USA) immediately following drug administration on the days depicted in Fig 1A and 1B. Behavioral testing was conducted during the light cycle for each mouse. Mice were habituated to the behavioral testing room one-hour prior to acclimate to fan noise. To reduce the effect of novelty on locomotor activity, mice were habituated to the locomotor boxes the day before the first experiment.
Adolescent C57BL/6 mice were repeatedly exposed to saline (SAL), 1.5 g/kg alcohol (ALC), 15 mg/kg caffeine (CAF), or caffeine-mixed alcohol (A+C) daily via intraperitoneal injection (n = 9–11 per group) for two weeks (male only, A) of four weeks (male and female, B). Locomotor activity was measured for 60 minutes directly following injection. Total distance traveled per session increased in animals exposed to caffeine-mixed alcohol over the exposure time for adolescent male mice (C). Adolescent male mice exposed to caffeine-mixed alcohol exhibited acute hyperlocomotion and significant locomotor sensitization between first and last exposure session measure in locomotor boxes over two weeks (D). Adolescent female animals sensitized more quickly and robustly than male mice (E) for animals exposed to caffeine-mixed alcohol over four weeks. Statistical significance was assessed by two-way, repeated measures ANOVA (time and treatment) followed by Bonferroni’s Multiple Comparison Test, *, p<0.05; **, p<0.01, ***, p<0.0005, ****, p<0.0001, ####, p<0.0001; data represented as mean ± SEM.
Adolescent male C57BL/6 mice (n = 6 per group) were administered water, caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by oral gavage for five days a week for four weeks (Fig 2). Locomotor activity was measured for 60 minutes in the locomotor activity boxes immediately following drug administration on the days depicted in Fig 2. Behavioral testing was conducted during the active/dark cycle for each mouse. Mice were habituated to the behavioral testing room one-hour prior to acclimate to fan noise. To reduce the effect of novelty on locomotor activity, mice were habituated to the locomotor boxes the day before the first experiment.
Male adolescent C57BL/6 mice were repeatedly exposed to exposed to water (H2O), 1.5 g/kg alcohol (ALC), 15 mg/kg caffeine (CAF) or caffeine-mixed alcohol (A+C), exposure by daily oral gavage (n = 6 per group) for 4 weeks for locomotor monitoring as depicted by the arrows. At the end of four weeks, animals were either perfused after one more drug administration (“IHC”) or subjected to behavioral tasks. Animals under “CPP” were subjected to cocaine conditioned place preference for cross-sensitization to cocaine reward. Animals in “SENS” were monitored for cocaine locomotor cross-sensitization. Natural reward consumption of saccharin was measured in “SACC” through four-hour limited-access, two-bottle choice between concentrations of saccharin (0.25, 0.5, 1.0, and 2.0 mM saccharin) and water for two days at each saccharin concentration.
Adolescent male C57BL/6 mice (n = 6 per group) were administered water, caffeine, alcohol, or caffeine-mixed alcohol via oral gavage or cocaine (15 mg/kg, i.p.), five days a week for four weeks during the animal’s dark/active cycle (Fig 2). Three days after the four week period of adolescent exposure, animals were once more exposed to their respective treatment and brains were collected 30 minutes later via transcardial perfusion as previously described by Engle et al, 2013  (Fig 2 “IHC”). Brains were fixed in a 4% paraformaldehyde solution for 24 hours before transfer into 30% sterile sucrose (Sigma) for one week for cryoprotection. Brains were embedded and frozen in Tissue-Tek® O.C.T. compound (VWR, Radnor PA, USA) in tissue molds (VWR) and 50 μm coronal sections were prepared using a cryostat (Leica Microsystems Inc., Buffalo Grove IL, USA). Staining was conducted on free-floating slices for ΔFosB positive cells using primary goat anti-ΔFosB antibody (sc-48-G, Santa Cruz Biotechnology, Dallas TX, USA), diluted 1:1000 and secondary Alexa-Fluor 594 donkey anti-goat antibody (A-11058, Life Technologies, Grand Island NY, USA), diluted 1:1000. Slices were mounted with VectaShield (Vector Laboratories, Burlingame CA, USA) mounting media on microscope slides (Fischer Scientific, Hampton NH, USA), fitted with coverglass (Fischer Scientific), and sealed with nail polish.
Images were acquired via confocal microscopy (Nikon A1) at 20x magnification using an oil immersion objective. Gain and exposure were standardized to slices from a water-treated animal for proper control throughout image capture. For each animal, two images were collected, one image from the left hemisphere and one from the right hemisphere for the brain region of interest. Images were processed using ImageJ software (National Institutes of Health) for the number of ΔFosB positive cells in the dorsal striatum and shell of the nucleus accumbens per image. Positive cells were identified as areas with a specific intensity and area compared to background, as identified through Image J analysis. The total area of analysis for each images = 403072 um2.
Adolescent male C57BL/6 mice (n = 8–12 per group) were administered water, caffeine, alcohol, or caffeine-mixed alcohol via oral gavage, five days a week for four weeks as previously described (Fig 2). The following week, mice were conditioned to cocaine in a conditioned place preference paradigm (CPP, Fig 2 “CPP”) . On day 1, mice were injected i.p. with saline and placed in a two-chamber conditioned place preference box (ENV-3013-2, Med Associates) to establish baseline preference the two chambers. Testing chambers contained unique tactile (wired mesh versus metal rod flooring) and visual (horizontal or vertical black and white striped wallpaper) cues for contextual usage to differentiate between the two chambers. Over the following eight conditioning days, mice received daily i.p. injection alternatively with saline or cocaine (1.5, 5, 15, or 30 mg/kg) and were confined for 30 minutes to either a cocaine-paired side or saline-paired side of the box in an unbiased approach. On the final day, saline was administered and the mice were placed in the CPP box in order to freely move between the two boxes for preference testing for 30 minutes (Fig 2). Preference was calculated as the difference in time spent in the cocaine-paired side between the pre- and post-conditioning tests. Mice that spent 70% of time in one side on the pre-conditioning day were excluded from the test. All conditioning was conducted during the dark/active cycle for each mouse.
Adolescent male C57BL/6 mice (n = 7–8 per group) were administered water, caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by oral gavage for five days a week for four weeks (Fig 2). Locomotor activity was measured for 60 minutes in the locomotor activity boxes on the first and final day of drug administration. Locomotor activity was measured as described previously for 60 minutes following habituation to the testing room during the animals’ dark/active cycle. Three days after final drug administration, animals were injected with 0.9% saline (i.p.) and placed in the locomotor boxes for baseline locomotor activity for 60 minutes. Two days after this baseline measurement (total of 5 days since last drug treatment), animals were injected with 15 mg/kg cocaine (i.p.) and placed in the locomotor boxes for 60 minutes for total locomotor activity measurement (Fig 2 “SENS”).
Natural reward was monitored through preference of sweet solution (saccharin) versus water in a four-hour, two bottle choice, drinking-in-the-dark paradigm  following adolescent exposure to drug solutions. Male adolescent C57BL/6 mice (n = 6–8 per group) were exposed to water or caffeine-mixed alcohol via oral gavage as described previously for four weeks in adolescence, shown in Fig 2. Upon final drug administration during the fourth week, animals were moved into single housing, double grommet cages for fluid consumption monitoring and to allow one weekend of acclimation to new cages. Three days after, saccharin solutions (0.25, 0.5, 1.0, 2.0 mM in reverse osmosis water) were prepared in 50 mL Falcon tubes, fitted with sippers, and distributed to the animals alongside a water control bottle during a four-hour, drinking-in-the-dark period to monitor saccharin consumption preference and volume (Fig 2 “SACC”) [30,31]. Bottles were added two hours into the dark cycle and removed four hours later, allowing behavioral testing during the animals’ active cycle. Weights of the bottles were measured to 0.1 gram. Each concentration was offered to the animals for two consecutive days before moving to the next concentration for total of eight days of drinking. The location of the water and saccharin bottles was reversed between days to prevent habit formation.
All data are presented as means ± standard error of the mean. The analysis of pharmacological drug effects over time was performed using one-way or two-way, repeated measures ANOVA for adolescent drug treatment and time, followed by a Bonferroni post-hoc test to determine statistically significant differences between groups using GraphPad Prism5 software (GraphPad Software, La Jolla, CA, USA). Student’s unpaired t-test was used for analyzing less than two groups using GraphPad Prism5.
We observed that adolescent mice exposed to caffeine-mixed alcohol or caffeine alone by i.p. injection (Fig 1A) displayed significant locomotor activity compared to water or alcohol alone as determined by two-way, repeated measures ANOVA (treatment: F3, 158 = 85, p<0.0001, time: F4, 158 = 7.74, p<0.0001), where we also observed a statistically significant interaction effect (interaction time x treatment: F12, 158 = 3.22, p<0.0004, Fig 1C). Comparison of locomotor activity after the first injection versus the last injection revealed that only caffeine-mixed alcohol exposure caused statistically significant locomotor sensitization (two-way, repeated measures ANOVA for time: F1, 67 = 16.70, p<0.0001, treatment: F3, 67 = 48.50, p<0.0001, interaction time x treatment: F3, 67 = 8.03, p<0.0001, Fig 1D). Female animals sensitized more quickly and robustly than male animals, although this difference was only apparent three weeks into testing (Fig 1B and 1E) as shown by two-way, repeated measures ANOVA for gender: F1, 17 = 5.51, p<0.0313, time: F4, 68 = 23.15, p<0.0001, and interaction time x gender: F4, 68 = 4.96 p<0.0014.
In order to increase the physiological relevance of the animal model while maintaining the ability to administer controlled amounts, we changed the exposure route from i.p. to oral gavage (Figs 2 and 3). We found that caffeine and caffeine-mixed alcohol significantly increased locomotor activity over four weeks of exposure (treatment: F4, 133 = 66.64, p<0.0001, time: F4, 133 = 0.67, p<0.6117, time x treatment F16, 133 = 2.13, p = 0.01, Fig 3A). In this model, we again observed that only adolescent mice exposed to caffeine-mixed alcohol showed significant locomotor sensitization versus caffeine alone between first and last drug exposure (two-way, repeated measures ANOVA for time: F3, 38 = 3.63, p = 0.06, treatment: F3, 38 = 35.18, p<0.0001, interaction time x treatment: F3, 38 = 7.82, p<0.0003 Fig 3B), although four weeks of exposure were necessary for these effects to be significantly different from the locomotor activity induced by caffeine alone.
Adolescent C57BL/6 mice were exposed to water (H2O), 1.5 g/kg alcohol (ALC), 15 mg/kg caffeine (CAF) or caffeine-mixed alcohol (A+C), exposure by daily oral gavage (n = 6 per group) for 4 weeks (Fig 2). Locomotor activity was measured for 60 minutes directly following injection. Mice exposed to caffeine-mixed alcohol showed acute hyperlocomotion and significant locomotor sensitization over the course of four weeks (A). Differences in first and last exposure demonstrate the increase in locomotor activity over the locomotor testing sessions (B). Statistical significance was assessed by two-way, repeated measures ANOVA (time and treatment) followed by Bonferroni’s Multiple Comparison Test, *, p<0.05; **, p<0.01, ***, p<0.0005, ****, p<0.0001, #, p<0.05; data represented as mean ± SEM.
The locomotor sensitization we observed in adolescent mice exposed to caffeine-mixed alcohol resembled the locomotor sensitization commonly observed upon chronic cocaine exposure . Chronic cocaine exposure is known to induce long-term increases in ΔFosB expression in the mesocortical and nigrostriatal dopaminergic pathways , thus we examined whether changes in ΔFosB expression occurred in the dorsal striatum and nucleus accumbens as a result of drug exposure (Fig 2). The shell of the nucleus accumbens was chosen (compared to nucleus accumbens core) as dopamine concentrations are known to preferentially increase in the shell following exposure to drugs of abuse . One-way ANOVA analysis of these data was statistically significant for both dorsal striatum (F4, 29 = 17.43, p<0.0001, Fig 4A, 4C and 4D) and nucleus accumbens (F4, 28 = 10.73, p<0.0001, Fig 4B, 4C and 4E) indicating that treatment in general affected ΔFosB expression. Post-hoc analysis with Bonferroni’s multiple comparison test revealed that mice exposed to cocaine, caffeine, alcohol, or caffeine-mixed alcohol exhibited a significant increase in the number of ΔFosB positive cells in the dorsal striatum compared to water controls. Interestingly, mice exposed to caffeine-mixed alcohol or cocaine during adolescence, but not alcohol or caffeine alone, exhibited increased ΔFosB expression in the nucleus accumbens versus water controls.
Considering the similarities between caffeine-mixed alcohol and cocaine with regard to locomotor sensitization, ΔFosB expression, and previous reports of caffeine induced sensitization of cocaine place preference [17,32], we next tested whether adolescent mice exposed to caffeine-mixed alcohol would show altered sensitivity to the rewarding properties of cocaine [32,33]. Mice were exposed to daily oral gavage injections of water, caffeine (15 mg/kg), alcohol (1.5 g/kg) or caffeine-mixed alcohol for four weeks during adolescence. Three days after final drug exposure, animals were subjected to cocaine conditioned place preference (Fig 2). Dose of 1.5, 5, 15, and 30 mg/kg were used to test preference exposed to caffeine-mixed alcohol in adolescence in separate cohorts of animals. Whereas animals exposed to water exhibited the strongest cocaine place preference to a dose of 15 mg/kg (Fig 5A) in accordance with that previously reported Hnasko et al., 2007 , caffeine-mixed alcohol exposed mice only showed significant place preference at 30 mg/kg of cocaine (two-way, repeated measures ANOVA for time: F1,13 = 13.47, p = 0.0023, treatment: F1,13 = 0.90, p = 0.3600, interaction time x treatment: F1,13 = 2.14, p = 0.1668, S1C Fig). No cocaine conditioned place preference was observed at 1.5 mg/kg for animals exposed to caffeine-mixed alcohol (S1A Fig) and no conditioning was observed in caffeine-mixed alcohol or water animals at 5 mg/kg cocaine (two-way, repeated measures ANOVA for time: F1,16 = 4.36, p = 0.053, treatment: F1,16 = 0.04, p = 0.8402, interaction time x treatment: F1,16 = 1.54, p = 0.2320, S1B Fig). Cocaine induced place preference at a dose of 15 mg/kg cocaine across all treatment groups except caffeine-mixed alcohol exposed animals, indicating that only caffeine-mixed alcohol exposed mice displayed desensitized place preference (two-way, repeated measures ANOVA for time: F1,29 = 28.17, p<0.0001, treatment: F3,29 = 0.70, p<0.5600, interaction time x treatment: F3,29 = 0.72, p<0.5501, Fig 5B).
We observed no difference in cocaine induced hyperlocomotion between water and caffeine-mixed alcohol exposed animals upon their first cocaine exposure during conditioning at any of the tested cocaine conditioning doses (S2A Fig). Additionally, there were no differences in 15 mg/kg cocaine induced locomotor activity during first conditioning session to cocaine between adolescent treatment groups (S2B Fig), suggesting that the attenuation in place preference observed in animals exposed to caffeine-mixed alcohol was not a result of alterations in locomotor response to cocaine. Adolescent exposure to caffeine-mixed alcohol also did not impact general locomotor activity during the pre-conditioning test day compared to water controls, although Bonferroni’s post-hoc analysis did show that caffeine exposed mice had significantly more locomotor activity than animals exposed to alcohol in adolescence (one-way ANOVA F3,29 = 4.976, p = 0.004, S2C Fig).
To investigate the effects of caffeine-mixed alcohol on cocaine locomotor cross-sensitivity, animals were exposed to 15 mg/kg cocaine after adolescent treatment (Fig 2). Exposure to caffeine alone increased both baseline (S3 Fig) and cocaine-induced increases in ambulation after adolescent t eatment (Fig 6), while exposure to water, alcohol, or caffeine-mixed alcohol did not (one-way ANOVA for baseline: F3,29 = 5.556, p = 0.0044, cocaine: F3,29 = 3.723, p = 0.0237).
We next investigated if exposure to caffeine-mixed alcohol during adolescence altered natural reward consumption and preference . To prevent satiation, saccharin solutions were chosen because of saccharin’s lack of caloric value compared to sucrose, which could inhibit drinking during the four-hour access period. Animals exposed to caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) during adolescence increased saccharin solution preference compared to animals exposed to water as observed by two-way, repeated measures ANOVA for adolescent treatment: F1,12 = 5.95, p = 0.031, saccharin concentration: F3,36 = 3.59, p = 0.023 (Fig 7A). Two-way, repeated measures ANOVA revealed significant differences in saccharin consumption as well, with Bonferroni post-hoc analysis indicating that animals exposed to caffeine-mixed alcohol consumed significantly larger quantities of 2 mM saccharin (Fig 7B, treatment: F1,12 = 7.62, p = 0.017, saccharin concentration: F3,36 = 16.13, p<0.0001). Analysis of cumulative saccharin intake revealed the same significant effect as (area under the curve shown in Fig 7C) as analyzed by student’s t-test.