Animals

Male Sprague-Dawley rats (Charles River) were received on either postnatal day 21 (P21) or P60 and double housed with food and water ad libitum. All experimental procedures were conducted during the light period of a 12-h light/dark cycle and were completed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Colorado Boulder.

Drugs

The nonselective adenosine receptor antagonist, caffeine, was purchased from Fisher Scientific (Waltham, MA). The dopamine D 2 receptor agonist, quinpirole ((−)-quinpirole hydrochloride), and cocaine hydrochloride were obtained from Sigma-Aldrich (St Louis, MO). All drugs, except caffeine, were dissolved in sterile-filtered physiological saline. Caffeine was dissolved in tap water.

Caffeine Consumption Procedure

Seven days after arrival, caffeine-consuming rats were given access to a single bottle containing caffeine in water (0.3 g/l) for 28 days (adolescent: P28–P55 or adult: P67–95; Figure 1). Age-matched control groups continued to receive water throughout the procedure. Caffeine and water consumption were monitored throughout the procedure. Following 28 days of caffeine exposure, the caffeine solution was replaced with water for the remainder of the experiment and behavioral testing was initiated at least 7 days after the last caffeine exposure. Thus, all behavioral testing and tissue collection were performed in the absence of caffeine between P62 and P82 or P101 and 121, periods corresponding to adulthood (Spear, 2000). Behavioral measures, tissue collection, and microdialysis studies were performed in separate cohorts of animals.

Figure 1 Caffeine consumption paradigm. (a) Time line of the model of adolescent caffeine consumption. Adolescent rats (P28–P55) consumed caffeine (0.3 g/l) and were tested in the absence of caffeine during adulthood (P62–P82). (b) Adolescent rats consumed an average of 27.34±1.128 mg/kg/day, although the dose diminished over the adolescent period (F 14,225 =130.2, p<0.0001). (c) There were no differences in the volume of fluid consumed by the caffeine-consuming rats (n=16) compared with the water controls (n=16). (d) Adolescent rats from both groups gained weight equivalently over the course of the consumption procedures. (e) Time line of the model of adult caffeine exposure. Adult rats (P67–P95) consumed caffeine (0.3 g/l) and were tested in the absence of caffeine (P101–P121). (f) The caffeine consumed remained stable over the procedure resulting in an average 23.78±0.24 mg/kg of caffeine consumed per day. (g) There are no differences in the volume of fluid consumed by the caffeine-consuming rats (n=10) compared with the water controls (n=10). (h) There were also no differences in weight gain in the caffeine-consuming rats compared with the water controls. PowerPoint slide Full size image

Locomotor Activity

Locomotor activity was conducted according to previously published procedures (Merritt and Bachtell, 2013). Briefly, animals underwent habituation to the locomotor chambers for 2 h on P62–65 (adolescent studies) or P101–104 (adult studies). On the following day, they were tested for cocaine- or quinpirole-induced locomotion in a single 4 h within-session escalating dose paradigm, where increasing doses of cocaine (vehicle, 2.5, 7.5 and 15 mg/kg, i.p.) or the dopamine D 2 receptor agonist, quinpirole, (vehicle, 0.1, 0.3 and 1.0 mg/kg, i.p.) were administered hourly. Locomotor activity was measured as the number of beam breaks during each hour of the testing period.

Place Conditioning

Place conditioning began 7 days following caffeine consumption (P62 for adolescent studies and P101 for adult studies) as described in the Supplementary Methods. Briefly, a three-phase procedure was conducted as follows: day 1—20 min pre-conditioning session, days 2–4—six 30 min conditioning sessions (0300 hours saline; 1500 hours cocaine) and day 5—20 min post-conditioning session. During the pre- and post-conditioning session, time spent in each compartment was recorded and the animals’ preference was determined by subtracting the time in the drug-paired compartment from the time in the saline-paired compartment. We used 7.5 and 15 mg/kg cocaine to condition a place preference. The 7.5 mg/kg cocaine was chosen because our previous studies have demonstrated that it does not reliably produce a place preference in all rats (Merritt and Bachtell, 2013), making this cocaine dose useful in identifying differences in the development of a place preference between water- and caffeine-consuming groups.

Sucrose Preference

A two-bottle choice paradigm was used to test sucrose preferences. Seven days after caffeine removal (P62), rats were habituated to drink water from two bottles for 3 days. The experimental procedures were conducted over the subsequent 4 days where consumption was measured between 1800 and 2200 hours, a period corresponding to the onset of the dark cycle. On each day, the bottles were replaced during this period as follows: day 1—water/water, day 2—water/0.5% sucrose, day 3—water/water, and day 4—water/0.05% sucrose. Total consumption and a preference ratio ((sucrose consumption/total consumption) × 100) were used for analysis.

Dopamine Microdialysis

On P62 (7 days following caffeine exposure), unilateral microdialysis cannula (CMA Microdialysis, Solna, Sweden) were implanted under halothane anesthesia (1–2.5%) into either the right or left NAc shell (relative to bregma: AP=+1.7, ML=±0.8, DV=−6.0) in a counterbalanced manner. Testing began ∼1 week after recovery from surgical procedures (∼P69). The evening before microdialysis testing, animals were transferred to the testing room and placed into separate plexiglass bowls containing bedding and ad libitum food and water. Microdialysis probes were inserted through the guide cannula and artificial CSF (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl, and 1.0 mM KCl) was perfused through the probes overnight using a BASi infusion pumps at a flow rate of 0.2 μl/min overnight. The flow rate was increased to 1.5 μl/min the next morning. Following 90 min of equilibration, three 20 min baseline samples were collected for the first hour of the experiment. Before the fourth sample tube was inserted, rats received cocaine (5 mg/kg, i.p.). This dose produces a submaximal increase in extracellular dopamine in the NAc and enables the detection of enhanced sensitivity for dopamine release. Samples were taken every 20 min for 3 h (total of nine samples) after injections, as previous studies indicate that cocaine-induced dopamine increases resolve within this time frame. Dopamine was quantified using an HPLC with electrochemical detection (ESA-Dionex, Sunnyvale, CA) in samples from animals whose cannulae were verified with cresyl violet staining post-mortem to be located within bounds of the desired region. One animal was removed from the study due to inaccurate placement.

Immunoblotting

Seven days following caffeine consumption (adolescent studies: P62 or adult studies: P101), rats were killed by rapid decapitation and bilateral 1 mm3 tissue punches were taken from chilled tissue slices containing the NAc and the caudate–putamen. Tissue punches were homogenized immediately and stored at −80 °C until protein levels were quantified by a Lowry protein assay. Samples (15 μg/well) from each animal were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. Blots were incubated with affinity-purified primary antibodies (see Supplementary Methods). All blots were stripped and re-probed for the loading control protein, β-tubulin. Secondary antibodies were detected by enhanced chemiluminescence (ECL film) and densitized. Blots were run with equal numbers of water-exposed control and caffeine-exposed samples per gel and loaded in an alternating manner. The results were quantified using ImageJ and the optical density for the proteins was normalized to β-tubulin.

Data Analysis

The effects of caffeine consumption on the various behavioral and neurobiological outcomes were analyzed separately for adolescent and adult consumption studies. Body weight and consumption data (mg/kg/day and ml/day) were analyzed using a two-way mixed-design ANOVA with consumption group (between) and days (within) as factors. Locomotor data were analyzed using a two-way mixed-design ANOVA with consumption group (between) and cocaine or quinpirole dose (within) as factors. Place conditioning data were analyzed using a two-way between-subject ANOVA with consumption group and cocaine dose as factors. Dopamine measures in the microdialysis experiments were analyzed with either an unpaired t-test to test for consumption group differences in basal dopamine or a two-way mixed-design ANOVA for cocaine-induced dopamine release with consumption group (between) and time (within) as factors. Finally, effects of caffeine exposure on protein expression were analyzed separately using an unpaired t-test. In all cases, significant interactions and main effects were followed by planned comparisons using one-way ANOVA or Bonferroni’s correction.