Journal: bioRxiv

Article Title: Chemogenetic activation of target neurons expressing insect Ionotropic Receptors in the mammalian central nervous system by systemic administration of ligand precursors

doi: 10.1101/2023.09.11.557081

Figure Lengend Snippet: ( A ) Robust expressions of IR84a and IR8a in iSPNs (A 2A R+), but not in dSPNs (D1R+) in Tg rats. A lentiviral vector expressing EGFP/IR84a and IR8a in a Cre-dependent manner was microinjected into the striatum of the Drd2 -Cre rats. Sections were triple-stained through immunohistochemistry for GFP, IR8a, and A 2A R, or GFP, IR8a, and D1R. Scale bar: 25 um. ( B ) Representative whole-cell recordings of the Lenti-FLEX-EGFP (control) or Lenti-FLEX-EGFP/IR84a-2A-IR8a (IR84a/IR8a) vector-treated striatal neurons of the Drd2 -Cre rats in response to PhAc. ( C-D ) Plot of changes in the membrane potential ( C ) and firing frequency ( D ) with addition of PhAc (0.1% w/v) in the control (n = 6) and IR84a/IR8a-expressing neurons (n = 4). Individual data points are overlaid *p < 0.05. Each white dot in the boxplot indicates the mean for the condition.

Article Snippet: Fixed brain were cut with a vibratome (VT1000S, Leica), and then sections (50-μm thick) were incubated with 10% normal donkey serum, and then mixtures of a primary antibodies for Cre (mouse, 1:200; MAB3120, Sigma-Aldrich), A 2A R (goat, 1:200; A2A-Go-Af700, Frontier Institute), and D1R (guinea pig, 1:200; D1R-GP-Af500, Frontier Institute) (for —figure supplement 1), or GFP (chicken, 1:1,000; ab13970, Abcam), IR8a (guinea pig, 1:1,000; ), and A 2A R, or GFP, IR8a, and D1R (goat, 1:200, D1R-Go-Af1000, Frontier Institute) (for ), or GFP and IR8a (for —figure supplement 1A).

Techniques: Plasmid Preparation, Expressing, Staining, Immunohistochemistry, Membrane

Journal: Nature Communications

Article Title: Purkinje cell dopaminergic inputs to astrocytes regulate cerebellar-dependent behavior

doi: 10.1038/s41467-023-37319-w

Figure Lengend Snippet: a A sagittal brain section from a Drd1a -tdTomato mouse. Five biological replicates were performed. Cx, cerebral cortex; Str, striatum; Th, thalamus; SNr, substantia nigra pars reticulata; Cb, cerebellum; GCL, granule cell layer; PCL, Purkinje cell layer; ML, molecular layer. Scale bar, 500 µm (left) and 50 µm (right). b Dual immunostaining of GFAP and tdTomato. Arrowheads indicate their colocalization. Five biological replicates were performed. Scale bar, 20 µm. c BG filled with biocytin followed by tdTomato immunostaining. Dotted outlines indicate their colocalization along the entire processes of BGs. Four biological replicates were performed. Scale bar, 20 µm. d D1R expression in Drd1a -tdTomato BGs (arrowheads). Four biological replicates were performed. Scale bar, 20 µm. e BG filled with biocytin followed by D1R immunostaining. Arrowheads indicate their colocalization. Four biological replicates were performed. Scale bar, 20 µm (left) and 2 µm (right, enlarged images). f Drd1a smFISH shows expression of Drd1a mRNAs at the border of the PCL and ML. Scale bar, 50 µm (inset, 10 µm). Three biological replicates were performed. g DAT1 expression in Drd1a -tdTomato sections. Arrowheads indicate BG soma; asterisks indicate PC soma. Four biological replicates were performed. Scale bar, 20 µm.

Article Snippet: Sections were permeabilized with 0.25% Triton X-100 for 2 h and blocked with 10% normal goat serum for 1 h. Sections were incubated at 4 °C overnight with blocking solution containing the following primary antibodies: mouse anti-GFAP (1:500, Sigma-Aldrich), rabbit anti-DsRed (1:2000, TaKaRa), mouse anti-RFP (1:1000, Rockland Immunochemicals), mouse anti-D1R (1:200, Sigma-Aldrich), rabbit anti-D1R (1:200, Abcam), mouse anti-DAT (1:200, Thermo Fisher Scientific), rabbit anti-GFP (1:2000, Abcam), chicken anti-GFP (1:2000, Abcam), mouse anti-calbindin D28K (CB) (1:500, Santa Cruz Biotechnology), rabbit anti-N terminal CYP2D6 (1:500; AV41675, Sigma-Aldrich), rabbit anti-C terminal CYP2D6 (1:500; PA5-79129, Thermo Fisher Scientific), mouse anti-TH (1:500, Sigma-Aldrich), rabbit anti-mCherry (1:2000, Abcam), rabbit anti-S100β (1:800, Sigma-Aldrich), guinea pig anti-c-fos (1:800, Synaptic Systems), or guinea pig anti-PV (1:800, Synaptic Systems).

Techniques: Immunostaining, Expressing

Journal: Nature Communications

Article Title: Purkinje cell dopaminergic inputs to astrocytes regulate cerebellar-dependent behavior

doi: 10.1038/s41467-023-37319-w

Figure Lengend Snippet: a Heat map of control and D1R cKO mice encountering the empty inverted cup (E) or the cup containing the first novel mouse 1 (S1) or S2 during the three-chamber social test. Minimal and maximum values indicate the relative accumulating activity in each area of the arena. b Time spent in the interaction with E, S1, or S2 ( n = 22 mice, Con: n = 12 mice, Drd1 cKO). Data are presented as mean ± SEM. Unpaired two-sided Student’s t test ( p = 0.0005, Con E vs. S1; p = 0.6135, Drd1 cKO E vs. S; p = 0.0010, Con S1 vs. S2; p = 0.4996, Drd1 cKO S1 vs. S2). c Phosphorylation levels of mTOR in the cerebellum of control and Drd1 cKO mice ( n = 6, each group). Data are presented as mean ± SEM. Mann–Whitney two-sided test ( p = 0.0019, Con vs. Drd1 cKO). d Phosphorylation levels of mTOR in cerebellar slices treated with vehicle (DMSO) for 1 h, SKF83822 for 1 h, or pretreated with SCH23390 followed by SKF83822 for 1 h ( n = 8, each group). Data are presented as mean ± SEM. Multiple unpaired two-sided Student’s t test ( p = 0.0020, Con vs. SKF, p = 0.0096, SKF vs. SCH). n.s., not significant.

Article Snippet: Sections were permeabilized with 0.25% Triton X-100 for 2 h and blocked with 10% normal goat serum for 1 h. Sections were incubated at 4 °C overnight with blocking solution containing the following primary antibodies: mouse anti-GFAP (1:500, Sigma-Aldrich), rabbit anti-DsRed (1:2000, TaKaRa), mouse anti-RFP (1:1000, Rockland Immunochemicals), mouse anti-D1R (1:200, Sigma-Aldrich), rabbit anti-D1R (1:200, Abcam), mouse anti-DAT (1:200, Thermo Fisher Scientific), rabbit anti-GFP (1:2000, Abcam), chicken anti-GFP (1:2000, Abcam), mouse anti-calbindin D28K (CB) (1:500, Santa Cruz Biotechnology), rabbit anti-N terminal CYP2D6 (1:500; AV41675, Sigma-Aldrich), rabbit anti-C terminal CYP2D6 (1:500; PA5-79129, Thermo Fisher Scientific), mouse anti-TH (1:500, Sigma-Aldrich), rabbit anti-mCherry (1:2000, Abcam), rabbit anti-S100β (1:800, Sigma-Aldrich), guinea pig anti-c-fos (1:800, Synaptic Systems), or guinea pig anti-PV (1:800, Synaptic Systems).

Techniques: Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Purkinje cell dopaminergic inputs to astrocytes regulate cerebellar-dependent behavior

doi: 10.1038/s41467-023-37319-w

Figure Lengend Snippet: Our findings suggest a model in which PCs synthesize DA through a non-canonical pathway and secrete it in an activity-dependent manner. The released DA binds D1Rs in BGs, inducing membrane depolarization and Ca 2+ signaling and driving membrane insertion of AMPAR GluA1 subunits. These actions may trigger glutamate release from BGs, which then enhances interneuron activity and reduces PF- and CF-PC synaptic transmission by potentially activating presynaptic GluRs. The increased inhibitory and the reduced excitatory inputs to PCs alter their firing frequency and pattern, ultimately impacting locomotor and social behaviors. BG, Bergmann glial cell; GC, granule cell; PC, Purkinje cell; MLI, molecular layer interneuron; CF, climbing fiber; PF, parallel fiber; DA, dopamine; D1R, D1 dopamine receptor; Glu, glutamate.

Article Snippet: Sections were permeabilized with 0.25% Triton X-100 for 2 h and blocked with 10% normal goat serum for 1 h. Sections were incubated at 4 °C overnight with blocking solution containing the following primary antibodies: mouse anti-GFAP (1:500, Sigma-Aldrich), rabbit anti-DsRed (1:2000, TaKaRa), mouse anti-RFP (1:1000, Rockland Immunochemicals), mouse anti-D1R (1:200, Sigma-Aldrich), rabbit anti-D1R (1:200, Abcam), mouse anti-DAT (1:200, Thermo Fisher Scientific), rabbit anti-GFP (1:2000, Abcam), chicken anti-GFP (1:2000, Abcam), mouse anti-calbindin D28K (CB) (1:500, Santa Cruz Biotechnology), rabbit anti-N terminal CYP2D6 (1:500; AV41675, Sigma-Aldrich), rabbit anti-C terminal CYP2D6 (1:500; PA5-79129, Thermo Fisher Scientific), mouse anti-TH (1:500, Sigma-Aldrich), rabbit anti-mCherry (1:2000, Abcam), rabbit anti-S100β (1:800, Sigma-Aldrich), guinea pig anti-c-fos (1:800, Synaptic Systems), or guinea pig anti-PV (1:800, Synaptic Systems).

Techniques: Activity Assay, Transmission Assay