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 quinpiroleinduced 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 reprobed 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 caffeineexposed 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,
= 20.50, p< 0.0001).
A
*
Preference Ratio
100
*
80
60
40
20
0
0
0.05
0.5
Sucrose Concentration (%)
B
Sucrose Consumed (ml)
24
20
Water
Caffeine
15
10
5
0
0.05
0.5
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
100.0 ± 8.6
107.5 ± 6.9
Adenosine A1
100.0 ± 2.9
102.0 ± 2.9
Adenosine A2
99.4 ± 7.3
Dopamine D1 100.0 ± 12.7
Dopamine D2
103.0 ± 4.8
100.0 ± 5.1
Dopamine Transporter
102.6 ± 3.1
100.0 ± 3.2
101.7 ± 4.6
Tyrosine Hydroxylase
100.0 ± 3.6
99.5 ± 3.5
DARPP-32
100.0 ± 2.6
Caudate Putamen: Adult
Adenosine A1
Adenosine A2
Dopamine D1
Dopamine D2
Dopamine Transporter
Tyrosine Hydroxylase
DARPP-32
100.0 ± 7.5
100.0 ± 6.4
100.0 ± 5.6
100.0 ± 2.5
100.0 ± 7.5
100.0 ± 6.8
100.0 ± 6.8
100.1 ± 7.5
106.4 ± 5.6
102.0 ± 6.7
105.2 ± 4.8
107.0 ± 19.0
98.9 ± 4.6
103.8 ± 6.8
Values are reported were normalized to -tubulin and are expressed as % of water control.