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.