Dopamine D1 Receptor

The NOR task was based on previously established protocols (Nelson et al., 2010, 2011 (link)). One day before the test day, animals received an acclimatisation session. The rats were placed individually into the arena for 1 h. On the following day, rats underwent a re-acclimatisation of 3 min to the arena. In experiments 1, 2 and 3, different groups of rats were injected with saline, 0.4 or 0.8 mg/kg of SKF81297 (s.c.) and returned to their home cage for 15 min. The rats were then given one 5 min sample phase in which the animals were allowed to explore two identical copies of the sample object. In experiment 3, rats were exposed to 5 min sample phase directly after the re-acclimatisation. The total time spent exploring the two identical objects was recorded. After a delay of 10 min (experiment 1) or 24 h (experiments 2 and 3), in which rats were return to their home cage, each rat was replaced for 3 min in the arena, which now contained a novel object and an identical copy of the object previously seen during the sampling phase. In every experiment, at the sampling phase the object exploration time was scored as the total over the full 5 min exposure to the objects. For the choice sessions, the time spent exploring the familiar and novel object was recorded over a total of 3 min. Because the preference for novel object, objects can decline rapidly, these exploration times were scored in three 1-min blocks.
Comparison between the systemic drug studies (experiments 1–3) provides the temporal resolution to distinguish effects at encoding versus retrieval in the NOR procedure. Since both encoding and retrieval were affected, the experiment 4 infusion study used the experiment 1 timeline (infusion before the sample phase and a 10 min retention interval) which does not distinguish effects on encoding and retrieval. Experiment 4 was run over a 6 day cycle in a within-subjects design to reduce the number of animals subjected to the cannulation procedure. On days 1, 3 and 5 rats were placed individually into the arena for 1 h. On days 2, 4 and 6, rats underwent a re-acclimatisation of 3 min to the arena before receiving one of three bilateral mPFC infusions 10 min before the sampling phase: saline, 0.05 μg (0.025 μg/side), 0.1 μg (0.05 μg/side) of the selective D₁ receptor agonist SKF81297. The order of the three infusions was counterbalanced using a Latin square design. During the 5 min sampling period the rats were exposed to two identical objects. After a delay of 10 min in which the rats were returned to their home cage, each rat was tested for 3 min in the arena containing a novel object and an identical copy of the object previously seen during the sampling phase. Again, the time spent exploring the familiar and novel object was recorded. In the course of the three sampling/choice sessions, three different object pairs were used, counterbalanced across the infusions conditions.

Samples were prepared in electrophoresis buffer in reducing and denaturing conditions (100 mM DTT, 2% SDS, 8% glycerol, 0.01% bromophenol blue and heated at 95 °C for 5 min). Denatured proteins (10–20 µg) were resolved on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. After being blocked for 1 h at room temperature (5% non-fat dry milk in PBS), membranes were incubated overnight at 4 °C with constant agitation with the primary antibodies against dopamine transporter (DAT) (Merk Millipore MAB369, 1:500, Darmstadt, Germany), noradrenaline transporter (NET) (MabTechnologies NET05-2, 1:1000, Neenah, WI, USA), tyrosine hydroxylase (TH) (Santa Cruz SC25269, 1:500, Dallas, TX, USA), dopamine D₁ receptor (D₁R) (Abcam AB78021, 1:500, Cambridge, MA, USA), dopamine D₂ receptor (D₂R) (Santa Cruz SC5303, 1:500, Dallas, TX, USA) or β-actin (Sigma-Aldrich A1978, 1:200,000, Sigma-Aldrich, St. Louis, MO, USA or Abcam AB8227, 1:10,000, Cambridge, MA, USA). After incubation with the fluorescent secondary antibodies (IRDyeTM 800 or Alexa Fluor® 680 conjugated) for 1 h at room temperature, the immunoreactive signal (integrated intensity values) was detected using the Odyssey infrared imaging system (LI-COR Biosciences) and quantified using Image Studio Lite 5.2 (LI-COR Biosciences). A standard pool of total homogenate was processed in the same gels and used as external reference sample. The immunoreactivity values were normalized for the β-actin signal. Antibody selection criteria was included in Additional file 2. Whole uncropped images of the original Western blots from which figures have been derived is shown in Additional file 3: Supplementary Fig. 1).

ELISAs were performed using 4 different neuronal antigens to detect autoantibodies in rabbit serum. Ninety-six well plates were coated overnight in 0.015 M carbonate/0.03 M bicarbonate (pH 9.6) buffer with each of the 4 antigens (Lysoganglioside, tubulin, human dopamine D1 receptor [D1R] antigen, and human dopamine D2 receptor [D2R]) as described previously [48 (link)]. ELISAs were then performed similar to what was described [49 (link)]. In short, washes were performed with PBS containing 0.05% Tween (PBS-Tween) five times and then blocked for 1 h. The wells were washed and sera from immunized or placebo rabbits were diluted and incubated overnight. After five washes, the samples were incubated with alkaline-phosphatase-labeled goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA). Then substrate and p-nitrophenyl phosphate were added. The optical density (OD) was measured at 405 nm. The results are expressed as the mean of triplicate wells. All ELISAs were validated with known positive and negative serum controls for each assay to maintain a standardized assay.