Journal: bioRxiv

Article Title: Chemogenetic ligands for translational neurotheranostics

doi: 10.1101/487637

Figure Lengend Snippet: ( A ) Compound 13 (C13) and Compound 22 (C22) structures. ( B ) C13 and C22 exhibit comparable DREADD affinity to clozapine (CLZ) with C13 showing ~2-fold greater affinity than C22. CLZ and C21 curves from are overlaid for comparison. ( C, D ) C13 selectively displaces [ 3 H]CLZ from AAV-DREADD sites at 10 nM. Representative images of sections collected from 3 different mice are displayed and quantified in D as mean ± SEM. Two-way ANOVA followed by Dunnett’s test, * p < 0.05 and ** p < 0.01 compared with the respective vehicle. ( E-H ) [ 3 H]C13 binds with greater selectivity than [ 3 H]CLZ to DREADDs in mouse and macaque brain tissue expressing AAV-hM3Dq and AAV-hM4Di respectively. ( I ) Intraperitoneal (IP) injection of [ 3 H]C13 readily enters the brain to binds to DREADDs. Representative images from 3 mice per condition. ( J-L ) JHU37107 (J07), JHU37152 (J52) and JHU37160 (J60) are high-affinity DREADD ligands. ( M ) Docking and molecular dynamics simulation of J60 in the ligand binding pocket of a hM4Di model. ( N-O ) J60 and J52 selectively displace [ 3 H]CLZ at a concentration of 1 and 10 nM from hM3Dq and hM4Di expressed in mouse brain sections (n = 3 mice per condition). ( P-Q ) J60 and J52 activate hM3Dq and hM4Di expressed in HEK293 cells with high potency (experiments performed 3 to 5 times). In all cases, data are represented as mean ± SEM.

Article Snippet: The following AAVs expressing hM4Di or hM3Dq fused to mCherry (or EGFP as a control) under the control of a hSyn promoter were used when indicated: hSyn-hM4D(Gi)-mCherry (Addgene: 50475-AAV8), hSyn-hM3D(Gq)-mCherry (Addgene: 50474-AAV8), hSyn-DIO-hM3D(Gq)-mCherry (Addgene: 44361-AAV8) and hSyn-EGFP (Addgene: 50465-AAV8).

Techniques: Expressing, Injection, Ligand Binding Assay, Concentration Assay

Journal: Journal of neurochemistry

Article Title: Regulating nociceptive transmission by VGluT2-expressing spinal dorsal horn neurons

doi: 10.1111/jnc.14588

Figure Lengend Snippet: A. Schematic representation of double-floxed Cre-dependent AAV8 vector expressing hM3D and mCherry downstream of the human synapsin promoter (hSyn). ITR, inverted terminal repeat. B and C. Low- and high-magnification confocal images show the distribution of mCherry-labeled (red) and NeuN-immunoreactive (green) neurons in the spinal dorsal horn of VGluT2-Cre mice 2 weeks after microinjection of the AAV-h3MD-mCherry vector. White dashed lines indicate the border of lamina II.

Article Snippet: We used 2 viral constructs, AAV8-hSyn-DIO-hM3Dq-mCherry and AAV8-hSyn-DIO-HA-KORD-IRES-mCitrine (#44361 and #65417, titer ≥ 4×10 12 vg/mL; University of North Carolina Vector Core, Chapel Hill, NC and Addgene, Cambridge, MA), to induce Cre-dependent expression of hM3D-mCherry and KORD-IRES-mCitrine in spinal dorsal horn neurons of VGluT2-Cre mice.

Techniques: Plasmid Preparation, Expressing, Labeling

Journal: Journal of neurochemistry

Article Title: Regulating nociceptive transmission by VGluT2-expressing spinal dorsal horn neurons

doi: 10.1111/jnc.14588

Figure Lengend Snippet: A. Representative recording traces show that bath application of 30 μM CNO depolarized and increased the firing frequency of lamina II neurons expressing hM3D-mCherry. Membrane potential is indicated on the left of individual recording traces. B. Quantification of the stimulatory effect of CNO on the firing rate of lamina II neurons expressing hM3D-mCherry (n = 15 neurons from 5 mice). C-E. Representative recording traces (C) and cumulative plots (D) and mean data with scattered plots (E) show the excitatory effect of 30 μM CNO on the frequency and amplitude of sEPSCs of lamina II neurons expressing hM3D-mCherry (n = 14 neurons from 4 mice). Data are expressed as mean ± S.E.M. * p < 0.05, ** p < 0.01 compared with the baseline control.

Article Snippet: We used 2 viral constructs, AAV8-hSyn-DIO-hM3Dq-mCherry and AAV8-hSyn-DIO-HA-KORD-IRES-mCitrine (#44361 and #65417, titer ≥ 4×10 12 vg/mL; University of North Carolina Vector Core, Chapel Hill, NC and Addgene, Cambridge, MA), to induce Cre-dependent expression of hM3D-mCherry and KORD-IRES-mCitrine in spinal dorsal horn neurons of VGluT2-Cre mice.

Techniques: Expressing