Supplementary Figure 3: Quinpirole induced GluN2B endocytosis is required for the effects of quinpirole on spines.
From: Age-dependent regulation of synaptic connections by dopamine D2 receptors
Primary hippocampal neurons (a, b, f–k) and cortical neurons (d, e) were treated with vehicle or quinpirole at DIV17 for 24 hrs. (a) Representative images of neurons stained for bassoon, synaptophysin, GluA1 or GluA2. (b) Quantification for (a). (c) The input–output relationship of EPSCNMDA recorded in hippocampal slices prepared from C57BL/6 mice intraperitoneally injected with D2R agonists (0.5 mg/kg of quinpirole or 10 mg/kg of bromocriptine). Since EPSCNMDA was comparable in quinpirole and bromocriptine treated slices (data not shown), data from these slices were pooled. n = 10 cells from 10 slices of 3 animals for each condition in (c). (d) Representative cropped immunoblots for phosphorylated GluN2B (Ser1303) and GluA1 (Ser845); the full–length images of western blots are shown in Supplementary Figure 7c. (e) Quantification for (d); n = 3 independent experiments. (f) Representative images of neurons transfected with the GFP–GluN2A construct. (g) Quantification for (f). (h) Representative images of GFP–GluN2B transfected neurons treated with 1 μM quinpirole alone or along with inhibitors for GluN2B (3 μM of ifenprodil or 1 μM of Ro 25–6891) or dynamin (20 μM). (i) Quantification for (h). (j) Representative images of neurons transfected with the Venus construct and treated with inhibitors as indicated. (k) Quantification of spine density for (k). For all images, n = 15 neurons for each condition. Scale bar, 20 μM. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.
