Journal: STAR Protocols

Article Title: Protocol to study the role of medial entorhinal cortex-basolateral amygdala circuit in context-induced retrieval of morphine withdrawal memory in mice

doi: 10.1016/j.xpro.2024.103542

Figure Lengend Snippet:

Article Snippet: Clozapine-n-oxide (CNO) , Selleckchem , #6887.

Techniques: Plasmid Preparation, Virus, Recombinant, Sterility, Saline, Software, Microinjection, Ointment, Purification, Microscopy

Journal: eNeuro

Article Title: A Novel Three-Choice Touchscreen Task to Examine Spatial Attention and Orienting Responses in Rodents

doi: 10.1523/ENEURO.0032-20.2021

Figure Lengend Snippet: DREADD expression in the SC across all DREADD-expressing animals. Expression is displayed across six slices, from bregma −5.52 mm as the most rostral, to bregma −6.60 mm as the most caudal slice. The color gradient on the SC represents the number of animals that had DREADD expression at each subregion of the SC: the darker the gradient at a subregion, the higher the number of animals that had DREADD expression in that subregion. Each schematic slice is accompanied by an immunohistochemistry photograph of the same slice from a representative animal. The images of the slices were taken from The Rat Brain Atlas in Stereotaxic Coordinates from .

Article Snippet: Animals were administered an intraperitoneal injection of the DREADD ligand CNO (Toronto Research Chemicals, C587520) at a dose of 3 mg/kg in 18% dimethyl sulfoxide (DMSO) in saline, or vehicle (18% DMSO, in saline), 20 min before testing.

Techniques: Expressing, Immunohistochemistry

Journal: Fundamental Research

Article Title: Engineering of rhesus monkey pluripotent stem cells for noninvasive survey and remote control after brain allotransplantation

doi: 10.1016/j.fmre.2024.03.003

Figure Lengend Snippet: Engineering and in vitro characterization of DREADD-rhPSCs for noninvasive survey and remote control. (a) Schematic representation of donor vector construction and strategy for knock-in mCherry or hM3Dq-mCherry expressing cassette into rhAAVS1 locus to generate mCherry- and DREADD-rhPSCs. SA, splicing acceptor; puro, puromycin resistance gene; CAG, cytomegalovirus immediate-early enhancer/chicken β-actin promoter; scissor mark, rhAAVS1 sgRNA targeting site; F1/R1 and F2/R2, genomic DNA PCR primer sets for genotyping; SB probe, probe used for Southern blot validation. (b) Representative immunofluorescence images of NANOG, OCT4 and mCherry staining in mCherry- and DREADD-rhPSCs. The nuclei are stained with Hoechst 33342 (HOE). Scale bars, 20 µm. (c) Representative immunofluorescence images of Ki67, SOX2, PAX6, N-Cadherin (N-Cad), NESTIN and mCherry staining in mCherry- and DREADD-rhPSC-derived cortical progenitors. Neuroepithelial organized into rosette-like structures are observed in both groups. Scale bars, 20 µm. (d) Quantification of positively labeled cells in (c). For mCherry group, Ki67 + , 84.98% ± 2.08%; SOX2 + , 96.76% ± 0.43%; PAX6 + , 95.90% ± 1.39%. For DREADD group, Ki67 + , 84.14% ± 2.39%; SOX2 + , 98.04% ± 0.69%; PAX6 + , 97.76% ± 0.43%. Data are presented as mean ± SEM, n = 5 for each group. (e) Representative immunofluorescence images of vGluT1, FOXG1, Tuj1 and mCherry staining in mCherry- and DREADD-rhPSC-derived cortical neurons. Scale bars, 20 µm. (f) Quantification of positively labeled cells in (e). For mCherry group, vGluT1 + , 93.10% ± 1.34%; FOXG1 + , 91.08% ± 0.94%; Tuj1 + , 98.16% ± 0.82%. For DREADD group, vGluT1 + , 92.88% ± 1.01%; FOXG1 + , 90.34% ± 1.42%; Tuj1 + , 97.54% ± 1.03%. Data are presented as mean ± SEM, n = 5 for each group. (g) Quantification of rest membrane potential (RMP) in 8-week-old glutamatergic neurons. For mCherry group, −37.33 ± 2.98 mV. For DREADD group, −42.83 ± 2.45 mV. Data are presented as mean ± SEM, n = 10 for each group, unpaired t- test, n.s., no significant difference. (h) Whole-cell patch current-clamp recordings in 10-week-old glutamatergic neurons derived from mCherry- and DREADD-rhPSCs showing representative changes in membrane potential (MP) upon CNO (10 µM) perfusion and washout. (i) Quantification of MP change in (h). For mCherry group, before vs. after CNO perfusion, −45.94 ± 2.64 mV vs. −45.18 ± 2.09 mV. For DREADD group, before vs. after CNO perfusion, −48.56 ± 1.29 mV vs. −41.96 ± 1.47 mV. Data are presented as mean ± SEM, n = 5 for each group, paired t- test, **, p < 0.01. (j) Whole-cell patch voltage-clamp recordings in 10-week-old glutamatergic neurons derived from mCherry- and DREADD-rhPSCs showing representative changes in sEPSC frequency and amplitude upon CNO perfusion and washout. Voltage was held at −70 mV. (k) Quantification of sEPSC frequency change in (j). For mCherry group, saline, 8.2 ± 0.92; CNO, 6.9 ± 1.30; washout, 9.2 ± 0.98. For DREADD group, saline, 5.4 ± 0.92; CNO, 14.3 ± 1.77; washout, 7.8 ± 1.18. Data are presented as mean ± SEM, n = 10 for each group, ordinary one-way ANOVA with Turkey test, **, p < 0.01, ***, p < 0.001. (l) Quantification of sEPSC amplitude change in (j). For mCherry group, saline, 53.39 ± 1.55 pA; CNO, 52.48 ± 2.23 pA; washout, 49.81 ± 1.23 pA. For DREADD group, saline, 52.16 ± 2.85 pA; CNO, 60.16 ± 1.44 pA; washout, 57.65 ± 2.11 pA. Data are presented as mean ± SEM, n = 10 for each group, ordinary one-way ANOVA with Turkey test, *, p < 0.05.

Article Snippet: For DREADD activation assays, 10 μL saline and 10 μL saline dissolved CNO (12 mM, MCE, HY-17366A) were sequentially supplied in perfused ACSF, with each solution having a total volume of 12 mL.

Techniques: In Vitro, Control, Plasmid Preparation, Knock-In, Expressing, Southern Blot, Biomarker Discovery, Immunofluorescence, Staining, Derivative Assay, Labeling, Membrane, Saline

Journal: Fundamental Research

Article Title: Engineering of rhesus monkey pluripotent stem cells for noninvasive survey and remote control after brain allotransplantation

doi: 10.1016/j.fmre.2024.03.003

Figure Lengend Snippet: Capturing the maturation and CNO-elicited responses in DREADD-rhPSC-derived neurons via [ 18 F]FDG-PET and EEG after rhesus monkey allotransplantation . (a) Representative coronal (COR) T1-weighted MR and [ 18 F]FDG-PET images of bilateral BA 9/46 in the rhesus monkey before transplantation (monkey #T1467001). PET was performed 10 min after i.v. injection of either saline or CNO (10 mg/kg). Dotted circles represent regions used for cell transplantation on both sides. Color scale, standard uptake value (SUV). R, right; L, left; H, head; F, foot. Scale bars, 10 mm. (b) Quantification of SUV ratio (SUVR, relative to cortex BA 10 control region) in (a). For saline group, 96.96% ± 0.89%. For CNO group, 96.64% ± 0.98%. Data are presented as mean ± SEM, n = 8 scans (obtained from 8 regions in BA 9/46 of monkeys #T1563105, #T1408071, #T1467001 and #T1403009 before transplantation), paired t- test. (c) Representative coronal and axial (AXI) [ 18 F]FDG-PET images in saline or CNO i.v. injection condition in a rhesus monkey transplanted with mCherry-rhPSC (right side)- and DREADD-rhPSC (left side)-derived cortical progenitors (monkey #T1408071, 6 months after transplantation). A, anterior; P, posterior. Scale bar, 10 mm. (d) Quantification of SUVR in (c) For saline group vs. CNO group in mCherry-neuron transplantations, 98.10 ± 0.76% vs. 98.78 ± 0.74%, n = 14 scan pairs (obtained from 2 regions in BA 9/46 of monkeys #T1563105, #T1408071). For saline group vs. CNO group in DREADD-neuron transplantations, 95.54% ± 0.48% vs. 101.53% ± 0.61%, n = 25 scan pairs (obtained from 4 regions in BA 9/46 of monkeys #T1563105, #T1408071, #T1467001 and #T1403009). Data are presented as mean ± SEM. Paired t- test was used for comparisons between saline-CNO treatment paired sessions, ****, p < 0.0001. Ordinary one-way ANOVA with Turkey test was used for comparisons between mCherry and DREADD groups, ## , p < 0.01. (e) Quantification of normalized SUVR ( SUV CNO − SUV saline SUV saline ) within saline-CNO treatment paired sessions. For nonoperative group, −0.33 ± 0.49%, n = 8 scan pairs (obtained from 4 regions in BA 9/46 of monkeys #T1563105, #T1408071, #T1467001 and #T1403009). For mCherry group, 0.71 ± 0.40%, n = 14 scan pairs (obtained from 2 regions in BA 9/46 of monkeys #T1563105, #T1408071). For DREADD group, 6.28±0.50%, n = 25 scan pairs (obtained from monkeys #T1563105, #T1408071, #T1467001 and #T1403009). Data are presented as mean ± SEM, ordinary one-way ANOVA with Turkey test, ****, p < 0.0001. (f) Representative axial T1-weighted MR image and time course of [ 18 F]FDG-PET images of bilateral BA 9/46 after CNO i.v. injection in a rhesus monkey transplanted with mCherry-rhPSC (right side)- and DREADD-rhPSC (left side)-derived cortical progenitors (monkey #T1563105). Scale bar, 10 mm. (g) Time course of normalized SUVR change in (f). For mCherry group, n = 2 regions (obtained from monkeys #T1563105 and #T1408071). For DREADD group, n = 4 regions (obtained from monkeys #T1563105, #T1408071, #T1467001 and #T1403009). Data plotted at the top panel are presented as mean (line) ± SEM (shade). Ordinary two-way ANOVA, ****, p < 0.0001. Data plotted at the bottom panel are individual SUVR change of both groups. (h) Schematic representation of the scalp electrode positions marked on the rhesus monkey head for EEG recording. 10 electrodes were placed on monkey scalp with medical EEG conductive paste according to 10–20 system. Reference electrodes were placed at Cz, A1 and A2 while the ground electrode was placed at FPz. Recording electrodes were placed at FP1, FP2, C3, C4, O1 and O2. (i) Normalized EEG power spectrum density (PSD) recorded by scalp electrodes close to the mCherry-graft (obtained from monkeys #T1563105 and #T1408071) and DREADD-graft (obtained from monkeys #T1563105, #T1408071, #T1467001 and #T1403009) in saline or CNO i.v. injection condition. (j) Quantification of the power in (i ) summed in high delta (2–4 Hz), theta (4–8 Hz), alpha (8–13 Hz) and beta (13–30 Hz) frequency bands. For mCherry group, before vs. after CNO perfusion, high delta, 16.15% ± 1.41% vs. 15.64% ± 1.25%; theta, 17.87% ± 2.21% vs. 16.06% ± 1.67%; alpha, 15.48% ± 2.34% vs. 14.94% ± 2.18%; beta, 30.16% ± 2.46% vs. 31.10% ± 2.14%; n = 5 records (obtained from monkeys #T1563105, #T1408071). For DREADD group, before vs. after CNO perfusion, high delta, 16.92% ± 0.48% vs. 14.83% ± 0.54%; theta, 17.27% ± 1.51% vs. 17.30% ± 1.25%; alpha, 14.50% ± 1.52% vs. 16.29% ± 1.37%; beta, 27.48% ± 0.86% vs. 34.51% ± 1.30%; n = 9 records (obtained from monkeys #T1563105, #T1408071, #T1467001 and #T1403009). Data are presented as mean ± SEM, paired t- test, *, p < 0.05, **, p < 0.01. (k) Representative EEG power spectra of FP1, FP2, C3, C4, O1, O2 channels in 1–30 Hz frequency band in saline or CNO i.v. injection condition in a rhesus monkey transplanted with mCherry-rhPSC (right side, close to FP2 electrode)- and DREADD-rhPSC(left side, close to FP1 electrode)-derived cortical progenitors (monkey #T1408071, 9 months after transplantation).

Article Snippet: For DREADD activation assays, 10 μL saline and 10 μL saline dissolved CNO (12 mM, MCE, HY-17366A) were sequentially supplied in perfused ACSF, with each solution having a total volume of 12 mL.

Techniques: Derivative Assay, Transplantation Assay, Injection, Saline, Control

Journal: Fundamental Research

Article Title: Engineering of rhesus monkey pluripotent stem cells for noninvasive survey and remote control after brain allotransplantation

doi: 10.1016/j.fmre.2024.03.003

Figure Lengend Snippet: Rhesus monkeys show no serious adverse events after allotransplantation and repeated DREADD activation. (a) Axial, coronal and sagittal (right/left SAG) T1-weighted and T2-weighted whole brain MR images of bilateral BA 9/46 (obtained from monkeys #T1563105, #T1408071 and #T1467001, 18 months after transplantation). Monkey #T1403009 is not suitable for MR imaging given he has several suspected subcutaneous metallic implants. Scale bars, 10 mm. (b) Schematic overview of in vivo study design showing the executing time point of CNO delivery, PET and EEG. (c) Representative 60 s EEG traces (80–250 Hz) of FP1, FP2, C3, C4, O1, O2 channels and corresponding time-frequency analysis (TFA, complex Morlet wave transformation) in saline or CNO i.v. injection condition. Naïve plot is obtained in a rhesus monkey with no surgical operation or transplantation. 9 m and 16 m plots are obtained in a rhesus monkey transplanted with mCherry-rhPSC (right side, close to FP2 electrode) and DREADD-rhPSC (left side, close to FP1 electrode)-derived cortical progenitors (monkey #T1408071, 9 and 16 months after transplantation). (d) Quantification of HFO frequency based on 80–250 Hz resting EEG of FP1, FP2, C3, C4, O1, O2 channels. For saline group vs. CNO group in experimental naïve monkeys, 1.00 ± 0.60 vs. 1.67 ± 0.67, n = 9 (obtained from 3 rhesus monkeys with no surgical operation or transplantation). For saline group vs. CNO group in DREADD-neuron transplanted monkeys, 2.11 ± 0.63 vs. 0.89 ± 0.54 in 9 m group and 1.22 ± 0.64 vs. 1.44 ± 0.60 in 16 m group; n = 9 for both groups (obtained from monkeys #T1563105, #T1408071 and #T1467001). Data are presented as mean ± SEM, ordinary one-way ANOVA with Turkey test. (e) Duration quantification of every HFO counted in (d). For saline group vs. CNO group in experimental naïve monkeys, 42.33 ± 3.41 ms vs. 39.00 ± 3.50 ms. For saline group vs. CNO group in DREADD-neuron transplanted monkeys, 35.21 ± 3.12 ms vs. 38.63 ± 5.05 ms in 9 m group and 37.91 ± 2.30 ms vs. 33.46 ± 2.14 ms in 16 m group. Data are presented as mean ± SEM, ordinary one-way ANOVA with Turkey test. (f) HFO duration summed in (e ) distributed in 10 ms bins.

Article Snippet: For DREADD activation assays, 10 μL saline and 10 μL saline dissolved CNO (12 mM, MCE, HY-17366A) were sequentially supplied in perfused ACSF, with each solution having a total volume of 12 mL.

Techniques: Activation Assay, Transplantation Assay, Imaging, In Vivo, Transformation Assay, Saline, Injection, Derivative Assay

Journal: Fundamental Research

Article Title: Engineering of rhesus monkey pluripotent stem cells for noninvasive survey and remote control after brain allotransplantation

doi: 10.1016/j.fmre.2024.03.003

Figure Lengend Snippet: The suicide system preserves CNO-elicited response of DREADD neurons in vivo . (a) Schematic overview of transplantation strategy for rhPSCs or rhPSC-derived cortical progenitors bearing suicide system, the time duration of daily GCV (10 mg/kg) i.v. drip, and the executing time point of PET, MR imaging and EEG. (b) Representative coronal and axial [ 18 F]FDG-PET images in saline or CNO i.v. injection condition in a rhesus monkey transplanted with HSVtk-rhPSC (right side)- and DREADD-rhPSC (left side)-derived cortical progenitors (monkey #T1467001, 14 months after transplantation). Scale bar, 10 mm. (c) Time course of [ 18 F]FDG-PET images of bilateral BA 9/46 in saline or CNO i.v. injection condition in a rhesus monkey (monkey #T1403009) transplanted with HSVtk-rhPSC (right side)- and DREADD-rhPSC (left side)-derived cortical progenitors before (16 months after transplantation) and 1 or 2 months after GCV treatment. Scale bar, 10 mm. (d) Quantification of SUVR in HSVtk-neuron transplantations. For saline group vs. CNO group before GCV treatment, 97.40% ± 0.98% vs. 102.52% ± 1.09%, n = 8 scan pairs (obtained from 2 regions in BA 9/46 of monkeys #T1467001 and #T1403009). For saline group vs. CNO group after GCV treatment, 95.28% ± 0.87% vs. 102.61% ± 1.42%, n = 4 scan pairs (obtained from 1 region in BA 9/46 of monkey #T1403009). Data are presented as mean ± SEM. Paired t- test was used for comparisons between saline-CNO treatment paired sessions, *, p < 0.05, ***, p < 0.001. Ordinary one-way ANOVA with Turkey test was used for comparisons between untreated and GCV treated groups. (e) Quantification of normalized SUVR within saline-CNO paired sessions. For the group before GCV treatment, 5.28% ± 0.83%, n = 8 scan pairs (obtained from 2 regions in BA 9/46 of monkeys #T1467001 and #T1403009). For the group after GCV treatment, 6.04% ± 1.23%, n = 4 scan pairs (obtained from 1 region in BA 9/46 of monkey #T1403009). Data are presented as mean ± SEM, unpaired t- test. (f) Normalized spectrum power recorded by scalp electrodes close to the HSVtk-graft before (obtained from monkeys #T1467001 and #T1403009) and after (obtained from monkey #T1403009) GCV treatment in saline or CNO i.v. injection condition. (g) Quantification of normalized spectrum power in (f) summed in high delta, theta, alpha and beta frequency bands. For the group before GCV treatment, before vs. after CNO perfusion, high delta, 17.66% ± 1.24% vs. 15.24% ± 0.67%; theta, 16.56% ± 1.20% vs. 17.95% ± 1.07%; alpha, 14.43% ± 1.17% vs. 18.03% ± 0.68%; beta, 26.93% ± 2.56% vs. 33.28% ± 1.61%; n = 8 records (obtained from monkeys #T1467001 and #T1403009). For the group after GCV treatment, before vs. after CNO perfusion, high delta, 19.74% ± 1.63% vs. 13.58% ± 1.30%; theta, 14.38% ± 0.33% vs. 15.49% ± 0.65%; alpha, 11.44% ± 0.98% vs. 15.60% ± 1.21%; beta, 27.51% ± 3.40% vs. 36.91% ± 3.82%; n = 8 records (obtained from monkey #T1403009). Data are presented as mean ± SEM, paired t- test, *, p < 0.05, **, p < 0.01. (h-i) Representative EEG power spectra of FP1, FP2, C3, C4, O1, O2 channels in 1–30 Hz frequency band in saline or CNO i.v. injection condition in a rhesus monkey (monkey #T1403009) transplanted with HSVtk-rhPSC (right side, close to FP2 electrode)- and DREADD-rhPSC (left side, close to FP1 electrode)-derived cortical progenitors before ((h), 16 months after transplantation) and 2 months after (i) GCV treatment.

Article Snippet: For DREADD activation assays, 10 μL saline and 10 μL saline dissolved CNO (12 mM, MCE, HY-17366A) were sequentially supplied in perfused ACSF, with each solution having a total volume of 12 mL.

Techniques: In Vivo, Transplantation Assay, Derivative Assay, Positron Emission Tomography-Magnetic Resonance Imaging, Imaging, Saline, Injection