Supplemental materials
METHODS
[3H]Raclopride binding to membranes from the midbrain and striatum
Membranes from the midbrain and striatum were prepared as described previously (Kramer et al. 2011). Then, 40 µg membrane proteins were suspended in binding buffer (50 mM Tris, pH 7.4) and incubated in the binding buffer containing a saturated concentration of [3H]raclopride (10 nM) for 3.5 hrs at 4°C. The non-specific binding was determined in the presence of (+)-butaclamol (4 µm). The reaction was terminated by the addition of 5 ml of ice-cold binding buffer, rapid filtration through polyethyleneimine-soaked GF/C filters using a brandel cell harvester, and two more 5 ml washes. The radioactivity on the filters was analyzed by a Beckman LS5801 liquid scintillation counter. The data were expressed as pmole/mg protein.
Western blot analyses of levels of subtypes of Gαi/o proteins in the midbrain
Since G proteins are membrane-bound, the expression of various types of Gαi/o proteins was determined from the membrane fractions prepared from the midbrain of control and amphetamine (AMPH) self-administering rats. Proteins (10 µg) were loaded on the SDS-PAGE. Gels were transferred electrophoretically to nitrocellulose membranes. Immunodetection was performed with the following antibodies: mouse anti-Gαi1 (Santa Cruz), rabbit anti-Gαi2 (Santa Cruz), mouse anti-Gαi3 (Santa Cruz), and rabbit anti-Gαo (Santa Cruz). Actin was used as a loading control. The band density was normalized to its respective actin level.
Co-localization of tyrosine hydroxylase (TH) and RGS2
Rats were perfused with 4% formaldehyde in PBS (pH 7.4). After perfusion, rat brains were sectioned into 30 µm free-floating cryosections with a microtome. Sections were permeabilized and blocked in the blocking buffer (10% normal goat serum in PBS containing 0.5% Triton X-100). Then slices were incubated sequentially with rabbit anti-TH (Santa Cruz) and mouse anti-RGS2 (Sigma). The corresponding secondary antibodies were goat anti-rabbit Alexa fluor 594 and goat anti-mouse Alexa fluor 488 (Invitrogen), respectively. Slices were mounted onto glass slides with ProLong Gold anti-fade reagent (Invitrogen). Images of cells displaying fluorescent signals were acquired on a Zeiss 710 laser scanning confocal microscope.
REFERENCES
Kramer, P. F., Christensen, C. H., Hazelwood, L. A., Dobi, A., Bock, R., Sibley, D. R., Mateo, Y. and Alvarez, V. A. (2011) Dopamine D2 receptor overexpression alters behavior and physiology in Drd2-EGFP mice. J Neurosci, 31, 126-132.
Figure S1. The expression levels of subtypes of Gαi/o proteins in the midbrain. (A) Representative blots for the levels of Gαi1, Gα2, Gαi3 and Gαo. (B) The average expression of subtypes of Gαi/o proteins in the midbrain from control and AMPH self-administering rats (N=5-6). There was no difference between groups in the expression of subtypes of Gαi/o proteins.
Figure S2. Surface dopamine D2/D3 receptor expression in the midbrain and striatum from control and amphetamine (AMPH) self-administering rats. The binding of D2/D3 receptor antagonist [3H]raclopride to dopamine D2/D3 receptors in the midbrain (A) or striatum (B) did not differ between the control and AMPH self-administering rats (N=6-8).
Figure S3. Colocalization of tyrosine hydroxylase (TH) and RGS2 in the ventral tegmental area (VTA) of a rat brain. VTA slices were immunostained with rabbit anti-TH and mouse anti-RGS2. TH and RGS2 were detected with goat anti-mouse AF488 and AF594, respectively. TH was co-localized with RGS2 in the dopaminergic neurons of the rat VTA.