Wild-type C57BL/6N mice were treated with THC (10 mg/kg as a single i.p. injection) or its vehicle. Four hours later, the striatum (St), cortex (Cx), hippocampus (Hc) and cerebellum (Cb) were …
Source data for THC impairs autophagy in the mouse striatum.
Primary striatal neurons from C57BL/6N mice were exposed for 24 hr to THC (0.75 µM) or HU-210 (10 nM), alone or in combination with hydroxychloroquine (0.1 mM), E64d (0.1 µM) and/or pepstatin A (10 …
Source data for THC impairs autophagy in primary striatal neurons.
Wild- type C57BL/6N mice were treated with temsirolimus (1 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single i.p. injection) or its …
Source data for Temsirolimus prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.
Wild-type C57BL/6N mice (n = 6–9 animals per group) were treated with temsirolimus (1 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single …
Wild-type C57BL/6N mice were given trehalose (10 g/L) or plain water ad libitum for 24 hr, and, subsequently, were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. (A) …
Source data for Trehalose prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.
Wild-type C57BL/6N mice (n = 6–10 animals per group) were given trehalose (10 g/L in drinking water) or plain water ad libitum for 24 hr, and, subsequently, with THC (10 mg/kg as a single i.p. inject…
(A–C) D1R-CB1R KO mice and CB1R-floxed control littermates were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. Panel A, Motor coordination (RotaRod test, time to fall …
Source data for Cannabinoid CB1 receptors located on D1R-MSNs, but not on glutamatergic neurons, are required for the THC-induced impairment of striatal autophagy and motor coordination in vivo.
Wild-type C57BL/6N mice (n = 5 animals per group) were treated with SR141716 (rimonabant; 3 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a …
(A) D1R-Cre mice and wild-type control littermates were injected stereotactically into the dorsal striatum with a CAG-DIO-dnRaptor rAAV, and left untreated for 4 weeks. Animals were subsequently …
Source data for mTORC1 and p62 in D1R-MSNs participate in the THC-induced impairment of motor coordination in vivo.
Drd1a-tdTomato;Drd2-EGFP mice were treated with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Immunofluorescence analysis of phosphorylated ribosomal protein S6 in the dorsal …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus, C57BL/6N, male) | Cnr1fl/fl;Drd1aCre | Monory et al., 2007; doi:10.1371/journal.pbio.0050269 | N/A | Conditional mutant mice in which the CB1 receptor gene (Cnr1) is absent from D1R (Drd1a)-expressing cells |
Strain, strain background (Mus musculus, C57BL/6N, male) | Drd1aCre | Lemberger et al., 2007; doi:10.1186/1471-2202-8-4 | N/A | Transgenic mice expressing Cre recombinase in D1R (Drd1a)-expressing cells |
Strain, strain background (Mus musculus, C57BL/6N, male) | Cnr1fl/fl;Neurod6Cre | Monory et al., 2006; doi:10.1016/j.neuron.2006.07.006 | N/A | Conditional mutant mice in which the CB1 receptor gene (Cnr1) is absent from dorsal telencephalic glutamatergic (Neurod6-expressing) neurons |
Strain, strain background (Mus musculus, C57BL/6N, male) | Drd1a-tdTomato;Drd2-EGFP | Suárez et al., 2014; doi:10.1016/j.biopsych.2013.05.006 | N/A | Transgenic mice expressing the tdTomato and EGFP reporter genes under the control of the Drd1a gene promoter and the Drd2 gene promoter, respectively |
Strain, strain background (Mus musculus, C57BL/6N, male) | C57BL/6N | Harlan Laboratories | RRID:MGI:5902763 | Wild-type mice |
Transfected construct (Homo sapiens) | Myc-Raptor (ΔCT) expression vector | Addgene Hara et al., 2002; doi:10.1016/s0092-8674(02)00833–4. Koketsu et al., 2008; doi:10.1152/ajpendo.00253.2007 | Plasmid #1859; RRID:Addgene_1859 | Vector backbone: pRK-5; construct generated by PCR-mediated deletion of1293 base pairs at the Raptor C-terminus |
Transfected construct (Homo sapiens) | HA-p62 expression vector | Addgene | Plasmid #28027; RRID:Addgene_28027 | Vector backbone: pcDNA4/TO |
Genetic reagent (Homo sapiens) | CAG-DIO rAAV expression vector | Klugmann et al., 2005; doi:10.1016/j.mcn.2004.10.002 Bellocchio et al., 2016; doi:10.1523/JNEUROSCI.1192–16.2016 | CAG-DIO rAAV Hybrid serotype 1/2 | Recombinant adeno-associated virus (rAAV) for Cre-driven transgene expression with a CAG promoter |
Biological sample (Mus musculus) | Primary striatal neurons | Harlan Laboratories (C57BL/6N mice) | C57BL/6N RRID:MGI:5902763 | In vitro cell cultures |
Antibody | Anti-LC3B (rabbit polyclonal) | Sigma-Aldrich | Cat. #L7543; RRID:AB_796155 | IF (1:300); WB (1:4000) |
Antibody | Anti-p62 (rabbit polyclonal) | Enzo Life Sciences | Cat. #BML-PW9860-0025; RRID:AB_2052149 | IF (1:250); WB (1:1000) |
Antibody | Anti-p62 (rabbit polyclonal) | Progen | Cat. #GP62-C; RRID:AB_2687531 | WB (1:1000) |
Antibody | Anti-LAMP1 (rabbit polyclonal) | Abcam | Cat. #ab25245 RRID:AB_449893 | IF (1:1000) |
Antibody | Anti-DARPP32 (mouse monoclonal) | BD Biosciences | Cat. #611520; RRID:AB_398980 | IF (1:700) |
Antibody | Anti-phospho-S6-S235/S236 (rabbit polyclonal) | Cell Signaling | Cat. #2211; RRID:AB_331679 | IF (1:300) |
Antibody | Anti-phospho-S6-S240/S244 (rabbit polyclonal) | Cell Signaling | Cat. #5364; RRID:AB_10694233 | IF (1:800) |
Antibody | Anti-c-Myc (mouse monoclonal) | Sigma-Aldrich | Cat. #11-667-149-001; RRID:AB_390912 | IF (1:500) |
Antibody | Anti-phospho-S6K-T389 (mouse monoclonal) | Cell Signaling | Cat. #9206; RRID:AB_2285392 | WB (1:1000) |
Antibody | Anti-total-S6K (rabbit polyclonal) | Cell Signaling | Cat. #9202; RRID:AB_331676 | WB (1:1000) |
Antibody | Anti-phospho-ULK1-S757 (rabbit polyclonal) | Cell Signaling | Cat. #6888; RRID:AB_10829226 | WB (1:1000) |
Antibody | Anti-phospho-ULK1-S555 (rabbit polyclonal) | Cell Signaling | Cat. #5869; RRID:AB_10707365 | WB (1:1000) |
Antibody | Anti-total-ULK1 (rabbit polyclonal) | Cell Signaling | Cat. #8054; RRID:AB_11178668 | WB (1:1000) |
Antibody | Anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Cat. #A5441; RRID:AB_476744 | WB (1:4000) |
Antibody | Anti-mouse monoclonal IgG (HRP-linked whole antibody) | GE-Healthcare Lifescience | Cat. #NA931; RRID:AB_772210 | WB (1:5000) |
Antibody | Anti-rabbit monoclonal IgG (HRP-linked whole antibody) | GE-Healthcare Lifescience | Cat. #NA934; RRID:AB_2722659 | WB (1:5000) |
Antibody | Goat anti-guinea pig IgG (H+L) (HRP-linked secondary antibody) | Invitrogen | Cat. #A18769; RRID:AB_2535546 | WB (1:5000) |
Antibody | Goat anti-mouse IgG (H+L) (cross-adsorbed, Alexa Fluor 488) | Invitrogen | Cat. #A-11001; RRID:AB_2534069 | IF (1:500) |
Antibody | Goat anti-mouse IgG (H+L) (cross-adsorbed, Alexa Fluor 594) | Invitrogen | Cat. #A-11005; RRID:AB_2534073 | IF (1:500) |
Antibody | Goat anti-mouse IgG (H+L) (cross-adsorbed, Alexa Fluor 647) | Invitrogen | Cat. #A-21235; RRID:AB_2535804 | IF (1:500) |
Antibody | Goat anti-rabbit IgG (H+L) (cross adsorbed, Alexa Fluor 488) | Invitrogen | Cat. #A-11008; RRID:AB_143165 | IF (1:500) |
Antibody | Goat anti-rabbit IgG (H+L) (cross adsorbed, Alexa Fluor 594) | Invitrogen | Cat. #A-11012; RRID:AB_2534079 | IF (1:500) |
Antibody | Goat anti-rabbit IgG (H+L) (cross adsorbed, Alexa Fluor 647) | Invitrogen | Cat. #A-21244; RRID:AB_2535812 | IF (1:500) |
Commercial assay or kit | Papain dissociation system (PDS) | Worthington | Cat. #LK 003153 | In vitro cell cultures |
Chemical compound, drug | Δ9-tetrahydro-cannabinol (THC) | THC Pharm GmbH | Dronabinol | In vivo experiments (10 mg/kg, i.p.);in vitro experiments (0.75 μM) |
Chemical compound, drug | Rimonabant (SR141716) | Cayman Chemical | Cat. #9000484 | In vivo experiments: (3 mg/kg, i.p.) |
Chemical compound, drug | Temsirolimus | LC Labs | Cat. #T-8040 | In vivo experiments (1 mg/kg, i.p.) |
Chemical compound, drug | Trehalose | Merck-Calbiochem | Cat. #90210 | In vivo experiments (10 g/L in drinking water) |
Chemical compound, drug | HU-210 | Tocris | Cat. #0966 | In vitro experiments (10 nM) |
Chemical compound, drug | Hydroxychloroquine | Merck | Cat. #509272 | In vitro experiments (0.1 mM) |
Chemical compound, drug | E64d | Enzo Life Sciences | Cat. #BML-PI107-0001 | In vitro experiments (0.1 μM) |
Chemical compound, drug | Pepstatin A | Enzo Life Sciences | Cat. #ALX-260–085 M005 | In vitro experiments (10 ng/ml) |
Software, algorithm | Graph Pad Prism 8.0 | GraphPad Software Inc | RRID:SCR_002798 | Descriptive analysis and statistics |
Software, algorithm | IBM SPSS | IBM Corporation | RRID:SCR_002865 | Statistical power analysis |
Software, algorithm | Image J | NIH | RRID:SCR_003070 | Western blot and immune-microscopy image analysis |
Software, algorithm | TCS-SP8 Leica Application Suite X, LASX | Leica | RRID:SCR_013673 | SP8 AOBS confocal microscopy image capture |
Software, algorithm | ACTITRACKUPG V2.7 | Panlab | Cat. #76–0610 | Motor activity analysis |
Other | DAPI stain | Invitrogen | Cat. #D1306; RRID:AB_2629482 | IF (1 µg/mL) |
Other | RotaRod LE8200 | Harvard Apparatus | Cat. #LE8200 (76–0237) | Motor coordination testing |
Other | IR actimeter (ActiTrack) | Panlab | Cat. #76–0127, #76–0131, #76–0134, #76–0125 | Motor activity testing |
Motor coordination (time to fall, s) | ||||||
---|---|---|---|---|---|---|
Raw Data | Vehicle | THC | ||||
Pre-treatment | Post-treatment | Post-treatment/Pre-treatment | Pre-treatment | Post-treatment | Post-treatment/Pre-treatment | |
93 | 101 | 1.08 | 113 | 71 | 0.63 | |
91 | 131 | 1.43 | 113 | 112 | 0.99 | |
79 | 122 | 1.54 | 101 | 105 | 1.05 | |
72 | 130 | 1.81 | 64 | 48 | 0.75 | |
100 | 143 | 1.44 | 80 | 80 | 1.00 | |
Mean | 87 | 125 | 1.46 | 94 | 83 | 0.88 |
SEM | 5.05 | 6.96 | 0.12 | 9.66 | 11.62 | 0.08 |
CI | 72.98 | 106.10 | 1.14 | 67.38 | 50.92 | 0.66 |
101.00 | 144.70 | 1.78 | 121.00 | 115.50 | 1.11 |