Figure legends
Figure 1 . Schematic representation of the experimental paradigm
performed in SERT−/− and SERT+/+rats. (A) The psychomotor effects of AMPH were assessed immediately
after AMPH injection and evaluated for 120 min. (B) AMPH
self-administration training was performed during 10 days, after which
the animals were subdivided into Short (ShA) or Long (LgA) access
groups. During the next 18 days the rats were allowed to self-administer
AMPH under either of these conditions. (C) 24 hrs after the final ShA or
LgA session rats were decapitated, and punches from the nucleus
accumbens core (cNAc) and shell (sNAc) sub regions (localized using the
Paxinos and Watson 2007 edition of the rat brain atlas) were collected
for gene expression analyses of the glutamatergic synapse.
Figure 2 . Psychomotor effects of AMPH in
SERT+/+ and SERT-/- animals. The
AMPH-induced and dose-dependent increase in locomotor activity was
stronger in SERT-/- vs SERT+/+ rats.
Data represent mean ± SEM distance moved. * p<0.05 versus
respective SERT+/+ vehicle; % p<0.05 versus
respective SERT−/− vehicle; # p<0.05 versus
amphetamine in SERT−/− rats; filled symbols versus
baseline (time 0).
Figure 3. AMPH self-administration in SERT+/+and SERT-/- animals. The escalation of AMPH intake
over the self-administration sessions was larger in rats under LgA than
under ShA conditions (A vs B). The daily AMPH intake was larger in
SERT-/- versus SERT+/+ rats under
LgA (B), but not ShA (A) conditions. Data are represented as mean ± SEM.
n.s.: not significant. * p≤0.05 (one-sided t-test) versus
SERT+/+.
Figure 4 . Interaction between SERT deletion and AMPH
self-administration on the vesicular glutamate transporter 1 (vGluT1) in
the cNAc and sNAc. Protein levels of vGluT1 in (A) cNAc and (B) sNAc are
expressed as percentages of SERT+/+-naive rats. Below
the graphs, representative immunoblots are shown for vGluT1 (60 kDa) and
β-actin (43 kDa) proteins in the homogenate of cNAc and sNAc. Histograms
represent the mean ± SEM of the following number of rats: naïve
(SERT+/+ n=6; SERT-/- n=6) ShA
(SERT+/+ n=14; SERT-/- n=13) and LgA
(SERT+/+ n=12; SERT-/- n=12).*p<0.05,***p<0.001 versus
SERT+/+-naive; #p<0.05,###p<0.001 versus
SERT-/--naive (Tukey’s multiple comparisons test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 5 . Interaction between SERT deletion and AMPH
self-administration on the glial glutamate transporter 1 (GLT-1) in the
cNAc and sNAc. Protein levels of GLT-1 in (A) cNAc and (B) sNAc are
expressed as percentages of SERT+/+-naive rats. Below
the graphs, representative immunoblots are shown for GLT-1 (62 kDa) and
β-actin (43 kDa) proteins in the homogenate of cNAc and sNAc. Histograms
represent the mean ± SEM of the following number of rats: naïve
(SERT+/+ n=6; SERT-/- n=6) ShA
(SERT+/+ n=14; SERT-/- n=13) and LgA
(SERT+/+ n=12; SERT-/- n=12).#p<0.05 versus
SERT-/--naïve; $$p<0.01
versus SERT-/--ShA (Tukey’s multiple comparisons
test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 6 . Interaction between SERT deletion and AMPH
self-administration on the NMDA receptor subunits in the cNAc and sNAc.
Data show GluN1 levels in (A) cNAc and (B) sNAc, GluN2A levels in (C)
cNAc and (D) sNAc, and GluN2B levels in (E) cNAc and (F) sNAc. In the
lower panel (G) representative immunoblots are shown for GluN2A (180
kDa), GluN2B (180 kDa), GluN1 (120 kDa) and β-actin (43 kDa) proteins in
the cNAc and sNAc homogenates of SERT+/+ and
SERT-/- rats exposed to AMPH. Results are expressed as
percentages of SERT+/+-naive rats. Histograms
represent the mean ± SEM of the following number of rats: naïve
(SERT+/+ n=6; SERT-/- n=6) ShA
(SERT+/+ n=14; SERT-/- n=13) and LgA
(SERT+/+ n=12; SERT-/- n=12).*p<0.05,**p<0.01,***p<0.001 versus
SERT+/+-naïve; #p<0.05,##p<0.01 versus
SERT-/--naïve; $p<0.05,$$p<0.01 versus
SERT-/--ShA (Tukey’s multiple comparisons test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 7 . Interaction between SERT deletion and AMPH
self-administration on the scaffolding protein SAP102 in the cNAc and
sNAc. Data show protein levels of SAP102 in the (A) cNAc and (B) sNAc
expressed as percentage of SERT+/+-naive rats. Below
the graphs, representative immunoblots are shown for SAP102 (102 kDa)
and β-actin (43 kDa) proteins in the homogenate of cNAc and sNAc.
Histograms represent the mean ± SEM of the following number of rats:
naïve (SERT+/+ n=6; SERT-/- n=6) ShA
(SERT+/+ n=14; SERT-/- n=13) and LgA
(SERT+/+ n=12; SERT-/- n=12).**p<0.01,***p<0.001 versus
SERT+/+-naïve (Tukey’s multiple comparisons test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 8 . Interaction between SERT deletion and AMPH
self-administration on the AMPA receptor subunits in the cNAc and sNAc.
Data show GluA1 levels in the (A) cNAc and (B) sNAc, and GluA2 levels in
the (C) cNAc and (D) sNAc. In the lower panel (E) representative
immunoblots are shown for GluA1 (108 kDa), GluA2 (108 kDa) and β-actin
(43 kDa) proteins in the cNAc and sNAc homogenates of
SERT+/+ and SERT-/- rats exposed to
AMPH. Results are expressed as percentages of
SERT+/+-naive rats. Histograms represent the mean ±
SEM of the following number of rats: naïve (SERT+/+n=6; SERT-/- n=6) ShA (SERT+/+ n=14;
SERT-/- n=13) and LgA (SERT+/+ n=12;
SERT-/- n=12). *p<0.05,**p<0.01 versus
SERT+/+-naïve; #p<0.05,##p<0.01,###p<0.001 versus
SERT-/--naïve; $$p<0.01
versus SERT-/--ShA (Tukey’s multiple comparisons
test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 9 . Interaction between SERT deletion and AMPH
self-administration on the scaffolding proteins SAP97 and GRIP in the
cNAc and sNAc. Data show SAP97 levels in the (A) cNAc and (B) sNAc, and
GRIP levels in the (C) cNAc and (D) sNAc. In the lower panel (E)
representative immunoblots are shown for GRIP (120 kDa), SAP97 (97 kDa)
and β-actin (43 kDa) proteins in the cNAc and sNAc homogenates of
SERT+/+ and SERT-/- rats exposed to
AMPH. Results are expressed as percentage of
SERT+/+-naive rats. Histograms represent the mean ±
SEM of the following number of rats: naïve (SERT+/+n=6; SERT-/- n=6) ShA (SERT+/+ n=14;
SERT-/- n=13) and LgA (SERT+/+ n=12;
SERT-/- n=12). *p<0.05,**p<0.01,***p<0.001 versus
SERT+/+-naïve; ##p<0.01
versus SERT-/--naïve;$$$p<0.001 versus
SERT-/--ShA (Tukey’s multiple comparisons test).
N=naïve, S=Amphetamine Short-Access; L= Amphetamine Long-Access
Figure 10 . Summary of the protein expression findings in the
nucleus accumbens shell and core of naïve SERT+/+ and
SERT-/- rats versus SERT+/+ and
SERT-/- rats exposed to ShA and LgA cocaine
self-administration.
cNAC=nucleus accumbens core, sNAC=nucleus accumbens shell,
ShA=Amphetamine Short-Access; LgA=amphetamine Long-Access,
vGlut1=vesicular glutamate transporter 1, GLT-1=glial glutamate
transporter 1, NMDA=NMDA receptor unit (e.g. GluN1),
Scaffolding=scaffolding protein (e.g. SAP102), AMPA=AMPA receptor
subunit
Supplementary figure 1 . Number of incorrect lever presses (A,
B) and timeout responses (C, D) during AMPH self-administration in
SERT-/- and SERT+/+ rats under ShA
and LgA conditions. No genotype differences were observed. Data are
represented as mean ± SEM. n.s.: not significant.
Supplementary figure 2 . Uncropped immunoblot related to the
expression levels of GluN2A (180 kDa), GluN2B (180 kDa), GluN1 (120
kDa), GRIP (135 kDa), SAP97 (97 kDa), SAP102 (102 kDa), GluA1 (108 kDa),
GluA2 (108 kDa), vGlut1 (67 kDa), GLT-1 (62 kDa) and β-Actin (43 kDa)
measured in the homogenate of the cNAc of SERT+/+ and
SERT-/- rats exposed to AMPH ShA and LgA protocol
presented in Fig 4, Fig 5, Fig 6, Fig 7, Fig 8, Fig 9. Samples were
loaded in three different gels as shown in panel A, B, C.
1= SERT+/+-naïve; 2= SERT+/+-ShA; 3=
SERT+/+-LgA; 4= SERT-/--naïve; 5=
SERT-/--ShA; 6= SERT-/--LgA.
Supplementary figure 3 . Uncropped immunoblot related to the
expression levels of GluN2A (180 kDa), GluN2B (180 kDa), GluN1 (120
kDa), GRIP (135 kDa), SAP97 (97 kDa), SAP102 (102 kDa), GluA1 (108 kDa),
GluA2 (108 kDa), vGlut1 (67 kDa), GLT-1 (62 kDa) and β-Actin (43 kDa)
measured in the homogenate of the sNAc of SERT+/+ and
SERT-/- rats exposed to AMPH ShA and LgA protocol
presented in Fig 4, Fig 5, Fig 6, Fig 7, Fig 8, Fig 9. Samples were
loaded in three different gels as shown in panel A, B, C.