We further confirmed that RGS2 protein exists in the midbrain dopaminergic neurons by immunocytochemistry

We further confirmed that RGS2 protein exists in the midbrain dopaminergic neurons by immunocytochemistry. by voltammetry. Using the antibody-capture [35S]GTPsubunit binding. Identification of RGS proteins that are specific for D2 receptor function Rabbit Polyclonal to RPL10L will improve our understanding of D2 receptor signaling and may lead to a future non-dopaminergic strategy for interference with DA transmission and drug abuse. As there are no selective pharmacological ligands that can differentiate D2 receptors from D3 receptors, results from experiments using quinpirole, a D2/D3 agonist, were applied to both D2 and D3 receptors. Here, we demonstrated that short-term AMPH self-administration reduced the ability of D2/D3 autoreceptors to regulate DA release and synthesis. Moreover, we showed for the first time that midbrain D2/D3 receptors Pirodavir were preferentially coupled to GAll animals were maintained according to the National Institutes of Health guidelines in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine. Self-Administration Rats were anesthetized and implanted with chronic indwelling jugular catheters as previously described (Liu fast-scan cyclic voltammetry (FSCV) was used to characterize D2/D3 autoreceptor function, DA transporter (DAT) activity, and DA release in the NAcc. Voltammetry experiments were conducted during the dark phase of the Pirodavir light cycle 18?h after commencement of the final AMPH self-administration session. A vibrating tissue slicer was used to prepare 400-m-thick coronal brain sections containing the NAcc, which were immersed in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4) and pH was adjusted to 7.4. Then the slice was transferred to the testing chambers containing aCSF at 32?C with a 1?ml/min flow rate. A carbon fiber microelectrode (100C200?M length, 7?M radius) and a bipolar stimulating Pirodavir electrode were placed into the core of the NAcc. DA release was evoked by a single electrical pulse (300?A, 4?ms, monophasic) applied to the tissue every 5?min. The extracellular DA level was recorded by applying a triangular waveform (?0.4 to +1.2 to ?0.4?V Ag/AgCl, 400?V/s). Once the extracellular DA level was stabilized, the amount of evoked DA release and a maximal rate of uptake (for 10?min at 4?C. The supernatants were centrifuged at 11?000?for Pirodavir 20?min. The resulting pellets were homogenized in 40 volumes of 10?mM NaHepes, pH 7.4, 1?mM MgCl2, 1?mM EGTA, and 1?mM DTT, and were centrifuged at 27?000?for 20?min. The resulting pellets were suspended in the same buffer at a protein concentration of 1 1.5?mg/ml and aliquots were frozen and stored at ?80?C. Stimulation of [35S]GTPfor 5?min. The radioactivity was detected on a Top-Count microplate scintillation counter (PerkinElmer). The non-specific binding was determined in the presence of unlabeled 10?M GTPfor 1?h. The supernatant was collected and stored at ?80C for further analyses of the cytosol-associated protein levels. The pellets were collected and resuspended by sonication in 20?mM Tris buffer (pH 8, containing 1?mM EDTA, 100?mM NaCl, 1% sodium deoxycholate, and 1?mM DTT and a cocktail of protease and phosphotase inhibitors) and lysed for 1?h at 4?C. The lysate was centrifuged at 100?000?for 60?min and the supernatants were collected for analyses of membrane-associated protein levels. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). Western Blot Analyses on Samples Prepared as Crude Homogenates, Cytosol Fractions, or Membrane Fractions from the Midbrain and the Striatum Quantitation of subtypes of GBonferroni test was used to compare group differences if necessary. A two-tailed Student’s controls. AMPH Self-Administration Resulted in Subsensitivity of D2/D3 Autoreceptors in Inhibition of Evoked DA Release To determine the ability of D2 autoreceptors to inhibit evoked DA release, increasing concentrations of the D2/D3 receptor agonist quinpirole (0.01C1?M) were added to the NAcc-containing slices to establish a doseCresponse curve. The peak in DA release was established at each concentration and expressed as the percent of control (Figure 2d). The IC50 for quinpirole was 22?nM in control rats and 463?nM in AMPH rats. A two-way ANOVA revealed a significant main effect of the quinpirole dose (F5,65=71.95, Bonferroni analysis indicated that quinpirole was less able to inhibit DA release at the 0.03, 0.1, and 0.3?M concentrations for AMPH self-administering rats as compared with controls, suggesting.