Supplemental Methods

Conditioned Place Preference

Animals were habituated to the room where the place conditioning procedures were performed for 1 hour the day before the pre-conditioning session. All testing and conditioning occurred in custom-built 3 compartment plexiglass chambers measuring 25 x 9 x 15 inches and equipped with 8 pairs of photobeams spaced 1 inch apart (Med

Associates Inc, St. Albans, VT). The two conditioning chambers were distinct in wall patterns and floor textures. During the pre-conditioning session, animals were placed in the center compartment of the apparatus and had access to the entire apparatus for 20 minutes. Their time spent in each compartment was recorded and the animals’ initial preference was determined. Animals displaying preferences greater that 80% for one compartment were excluded from the experiment. Conditioning was performed in an unbiased counterbalanced fashion where animals received three 30 min saline conditioning sessions and three 30 min cocaine (7.5 mg/kg or 15 mg/kg, i.p.) conditioning sessions. Saline conditioning occurred between 0800–1100 hrs, while cocaine conditioning occurred between 1500–1700 hrs. The 7.5 mg/kg cocaine dose was chosen because, unlike the 15 mg/kg dose, previous studies in our lab have demonstrated that it does not reliably produce a place preference in all rats. Therefore, this cocaine dose was ideal to identify potential differences in the development of a place preference between the two groups. After the conditioning sessions, a post-conditioning test was conducted where animals were placed in the central compartment of the apparatus for 20 minutes. Preference was determined as time spent in the drug compartment minus time spent in the saline compartment (conditioned place preference (CPP) score). Locomotor testing

Following at least 7 days withdrawal from caffeine exposure, cocaine- and quinpirole- induced locomotion was recorded in plexiglass chambers (San Diego Instruments, San

Diego CA) measuring 16x16x15 inches with 16 pairs of photobeams spaced 1 inch apart on both the x- and y-axis. All locomotor testing occurred in complete darkness. On the test day, animals underwent habituation to the locomotor chambers for 2 hours.

Following habituation the animals were treated with increasing doses of cocaine (vehicle,

2.5 mg/kg, 7.5 mg/kg, 15 mg/kg) each hour. Likewise, we examined how caffeine exposure affects D2 agonist (quinpirole)-induced locomotor activity using a similar procedure. Following a 2 hr habituation period, animals were treated with increasing doses of cocaine (vehicle, 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg) each hour. Total locomotor activity was measured as the number of beam breaks during each hour of the testing period.

Immunoblotting

On P62 or P102 (7 days following caffeine exposure in adolescents and adults, respectively), rats were killed by rapid decapitation and bilateral 1 mm3 tissue punches were taken from chilled tissue slices containing the NAc shell and the caudate putamen.

Tissue punches were homogenized immediately and stored at -80ºC 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 that probe for adenosine A1 (1:1000 Calbiochem/EMD Millipore, Billerica, MA), adenosine A2A (1:2000 Millipore,

Billerica, MA) receptors, dopamine D1 (1:1000 Millipore, Billerica, MA), dopamine D2 receptors (1:2000 Millipore, Billerica, MA), dopamine transporter (DAT, 1:2000

Millipore, Billerica, MA), tyrosine hydroxylase (1:1000 Cell Signaling, Beverly, MA), and DARPP-32 (1:1000 Cell Signaling, Beverly, MA). All blots were stripped and re- probed for the loading control protein, β-Tubulin (1:1000 Millipore, Billerica, MA).

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 fashion. The results were quantified using ImageJ and the optical density for the proteins was normalized to β-Tubulin. Supplemental Results

Sucrose Preference in Animals Exposed to Caffeine During Adolescence

Chronic exposure to caffeine during adolescence has no effect on sucrose preference

(Supplemental Figure 1A and 1B). A repeated measures 2-way ANOVA found no sucrose X treatment interaction (F2,24 = 1.27, p= 0.30) and no significant main effect of treatment (F1,24 = 0.22, p= 0.65) at the 2 hr time point (supplementary figure 1A).

However, there was a significant main effect of sucrose (F2,24 = 15.54, p< 0.0001) at the 2 hr time point. Similarly, at the 16 hr time point (supplementary figure 1B) there was no sucrose X treatment interaction (F2,24 = 0.74, p= 0.49), and no significant main effect of treatment (F1,24 = 1.86, p= 0.20). Again, there was a significant main effect of sucrose (F2,

24 = 20.50, p< 0.0001).

A B ) 100 * * l 20 m Water (

o i d t 80 Caffeine e a 15 R m

u

e 60 s c n

n 10 o e r 40 C

e f e e s

r 5 o r

P 20 c u

0 S 0 0 0.05 0.5 0.05 0.5 Sucrose Concentration (%) Sucrose Concentration (%)

Supplemental Figure 1. Rats exposed to caffeine during adolescence do not differ from water controls in sucrose preference or consumption. A) Animals from both water and caffeine consumption groups preferred both a low and high sucrose concentration compared to water (* Bonferonni post hoc, p< 0.0001). B) Volume of sucrose consumed was also not different between the water- and caffeine-consuming animals. Supplemental Table 1: Effects of adolescent and adult caffeine consumption on protein expression in the caudate putamen Control (n=8) Caffeine (n=8) Caudate Putamen:Adolescent

Adenosine A1 100.0 ± 8.6 107.5 ± 6.9 Adenosine A2 100.0 ± 2.9 102.0 ± 2.9 Dopamine D1 100.0 ± 12.7 99.4 ± 7.3 Dopamine D2 100.0 ± 5.1 103.0 ± 4.8 Dopamine Transporter 100.0 ± 3.2 102.6 ± 3.1 Tyrosine Hydroxylase 100.0 ± 3.6 101.7 ± 4.6 DARPP-32 100.0 ± 2.6 99.5 ± 3.5

Caudate Putamen: Adult

Adenosine A1 100.0 ± 7.5 100.1 ± 7.5 Adenosine A2 100.0 ± 6.4 106.4 ± 5.6 Dopamine D1 100.0 ± 5.6 102.0 ± 6.7 Dopamine D2 100.0 ± 2.5 105.2 ± 4.8 Dopamine Transporter 100.0 ± 7.5 107.0 ± 19.0 Tyrosine Hydroxylase 100.0 ± 6.8 98.9 ± 4.6 DARPP-32 100.0 ± 6.8 103.8 ± 6.8 Values are reported were normalized to -tubulin and are expressed as % of water control.