Journal: International Journal of Neuropsychopharmacology

Article Title: Involvement of the Striatal Medium Spiny Neurons of the Direct Pathway in the Motor Stimulant Effects of Phencyclidine

doi: 10.1093/ijnp/pyv134

Figure Lengend Snippet: Effects of acute administration of phencyclidine (PCP) on dopamine and adenosine 3’,5’-cyclic monophosphate activated phosphoprotein of 32 kDa (DARPP-32) phosphorylation in D32 F/F D1RCre + and D32 F/F D2RCre + mice. (A) D32 F/F D1RCre + and (B) D32 F/F D2RCre + conditional knockout mice and (A) D1RCre and (B) D2RCre control mice were treated with one injection of vehicle or PCP (3mg/kg) and killed by decapitation after 30min. DARPP-32 phosphorylation on Thr34 was determined by Western blotting as described in Methods. Upper panels are representative Western blots showing phosphorylated DARPP-32 (top) and total DARPP-32 (bottom). Note the reduction in total DARPP-32 produced by genetic inactivation in (A) D1R- or (B) D2R-expressing MSNs. The increase in phosphorylated DARPP-32 induced by PCP is abolished in (A) D32 F/F D1RCre + mice, but still present in (B) D32 F/F D2RCre + mice. Lower panels are means ± standard error of the mean (n = 5–8/group) of data expressed as percent of (A) D1RCre or (B) D2RCre mice injected with vehicle. * p < 0.05 and ** p < 0.01 vs. vehicle-treated mice, same genotype; ## p < 0.01 vs. vehicle-treated mice of respective control genotype (two-way ANOVA, followed by Fisher’s post hoc comparison).

Article Snippet: The membranes were incubated 40min in 5% milk blocking solution and then incubated with antibodies against total DARPP-32, or phospho-Thr34-DARPP-32 (PhosphoSolutions).

Techniques: Phospho-proteomics, Knock-Out, Control, Injection, Western Blot, Produced, Expressing, Comparison

Journal: International Journal of Neuropsychopharmacology

Article Title: Involvement of the Striatal Medium Spiny Neurons of the Direct Pathway in the Motor Stimulant Effects of Phencyclidine

doi: 10.1093/ijnp/pyv134

Figure Lengend Snippet: Effects of repeated administration of phencyclidine (PCP) on dopamine and adenosine 3’,5’-cyclic monophosphate activated phosphoprotein of 32 kDa (DARPP-32) phosphorylation in D32 F/F D1RCre + and D32 F/F D2RCre + mice. (A) D32 F/F D1RCre + and (B) D32 F/F D2RCre + conditional knockout mice and (A) D1RCre and (B) D2RCre control mice were treated for 8 days (one injection/day) with vehicle or PCP (3mg/kg) and killed by decapitation 30min after the last injection. DARPP-32 phosphorylation on Thr34 was determined by Western blotting as described in Methods. Upper panels are representative Western blots showing phosphorylated DARPP-32 (top) and total DARPP-32 (bottom). Note the reduction in total DARPP-32 produced by genetic inactivation in (A) D1R- or (B) D2R-expressing MSNs. The increase in phosphorylated DARPP-32 induced by PCP is abolished in (A) D32 F/F D1RCre + mice, but still present in (B) D32 F/F D2RCre + mice. Lower panels are mean ± standard error of the mean (n = 5–8/group) of data expressed as percent of (A) D1RCre or (B) D2RCre mice injected with vehicle. * p < 0.05 and ** p < 0.01 vs. vehicle-treated mice, same genotype; ## p < 0.01 vs. vehicle-treated mice of respective control genotype (two-way ANOVA, followed by Fisher’s post hoc comparison).

Article Snippet: The membranes were incubated 40min in 5% milk blocking solution and then incubated with antibodies against total DARPP-32, or phospho-Thr34-DARPP-32 (PhosphoSolutions).

Techniques: Phospho-proteomics, Knock-Out, Control, Injection, Western Blot, Produced, Expressing, Comparison

Journal: Scientific Reports

Article Title: New MiniPromoter Ple389 ( ADORA2A ) drives selective expression in medium spiny neurons in mice and non-human primates

doi: 10.1038/s41598-024-79004-y

Figure Lengend Snippet: Three MiniPromoters delivered by ICV injection in mice showed co-labeling with a medium spiny neuron-specific marker in the striatum. MiniPromoters, driving EmGFP in rAAV-PHP.B, were delivered by ICV injection into the lateral ventricle of adult mice, and harvested 4 weeks later. High-power view (left) and quantification of co-localization (right) are shown for Ple385 ( GPR6 ), Ple389 ( ADORA2A ), and Ple391 ( DRD2 ) following double immunostaining of striatal cells for GFP and DARPP32, a marker for striatal MSNs. A considerable number of GFP-labelled cells in the striatum co-expressed DARPP32 immunoreactivity after ICV injections of the three MiniPromoters, confirming specific MSNs expression. EmGFP, emerald-green fluorescent protein; rAAV9-PHP.B, recombinant adeno-associated virus packaged in capsid 9 variant PHB-B. Green, anti-GFP; red, anti-DARPP32. Scale bars, 100 µm.

Article Snippet: Rat anti-DARPP32 , R&D System (Minneapolis, MN) , MAB4230 , Mice (ICV) , 1:1000.

Techniques: Injection, Labeling, Marker, Double Immunostaining, Expressing, Recombinant, Virus, Variant Assay

Journal: Scientific Reports

Article Title: New MiniPromoter Ple389 ( ADORA2A ) drives selective expression in medium spiny neurons in mice and non-human primates

doi: 10.1038/s41598-024-79004-y

Figure Lengend Snippet: Source and concentrations of antibodies and probes.

Article Snippet: Rat anti-DARPP32 , R&D System (Minneapolis, MN) , MAB4230 , Mice (ICV) , 1:1000.

Techniques: Concentration Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Environmental Enrichment Alters Nicotine-Mediated Locomotor Sensitization and Phosphorylation of DARPP-32 and CREB in Rat Prefrontal Cortex

doi: 10.1371/journal.pone.0044149

Figure Lengend Snippet: Ratio of phosphorylated Thr 34 and Thr75 to total DARPP-32 in different brain regions in EC and IC rats after systemic nicotine administration.

Article Snippet: Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 55–65 min at 150 V, and transferred to Immobilon-P transfer membranes (Cat # IPVH00010, 0.45 μm pore size; Millipore Co., Bedford, MA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS with 20% methanol) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories Ltd., Hercules, CA) for 110 min at 75 V. Transfer membranes were then incubated with blocking buffer (5% dry milk powder in PBS containing 0.5% Tween 20) for 1 h at room temperature and then incubated overnight at 4°C in blocking buffer with primary antibodies against the following proteins: DARPP32 (1∶2000, 374-DARPP, PhosphoSolutions, Aurora, CO), pDARPP32 Thr34 (1∶1000, p1025–34, PhosphoSolutions, Aurora, CO), pDARPP32 Thr75 (1∶1000, p1025–75, PhosphoSolutions, Aurora, CO), CREB (1∶1000, 9104, Cell Signaling, Danvers, MA), pCREB (1∶1000, 9196L, Cell Signaling, Danvers, MA), respectively.

Techniques: