Journal: bioRxiv

Article Title: A Cultured Sensorimotor Organoid Model Forms Human Neuromuscular Junctions

doi: 10.1101/2021.03.03.433128

Figure Lengend Snippet: a, Live neuronal labeling by expression of hSYN ::EGFP delivered by an AAV9 vector. Scale bar, 100 µm. b, Quantification of neurite area as fold change of initial, from three to nine weeks in culture. Line indicates mean and shaded area indicates S.E.M.(effect of time on neurite area, n =6 organoid cultures obtained from 3 separate differentiations, linear model, F=8.743, P=1.3×10 -10 , individual comparisons: TDP-43 pair: F=37.040, P=2.0×10 -8 ; SOD1 pair: F=16.3725, P=1.0×10 -4 ; PFN1 pair: F=18.639, P=3.6×10 -5 ; interaction between time and iPSC line on neurite area, n =6 organoid cultures obtained from 3 separate differentiations, linear model, F=0.562, P=0.985). c, Normalized percentage of NMJ innervation at three months in culture (normalized to the paired isogenic controls). Bars indicates mean and S.E.M. ( n =6 organoid cultures obtained from 3 separate differentiations identified by colors, Univariate ANOVA: F=5.938, P=6.3×10 -4 ; separate two-tailed t-test; TDP-43 pair: P=0.560; SOD1 pair: P=0.010; PFN1 pair: P=0.047). d, Normalized area of the innervated NMJs at three months in culture (normalized to the paired isogenic controls). Bars indicate mean and S.E.M. ( n =6 organoid cultures obtained from 3 separate differentiations identified by colors, Univariate ANOVA: F=3.001, P=0.026; separate two-tailed t-test; TDP-43 pair: P=0.009; SOD1 pair: P=0.203; PFN1: P=0.659). e, Example of the reduction in NMJ area observed in the TDP-43 +/G298S iPSC line compared to the isogenic control. Scale bar, 100 µm.

Article Snippet: Organoid cultures were transduced with an hSYN ::EGFP AAV9 viral vector (a gift from Bryan Roth (Addgene viral prep # 50465-AAV9; http://n2t.net/addgene:50465 ; RRID:Addgene_50465) at week 2 of culture.

Techniques: Labeling, Expressing, Plasmid Preparation, Two Tailed Test, Control

Journal: Experimental Neurobiology

Article Title: AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus

doi: 10.5607/en.2017.26.6.350

Figure Lengend Snippet: AAV-hALDH1L1-Cre virus test in primary cultured system. (A) Map of AAV vectors expressing Cre recombinase under h ALDH1L1 and CaMKIIa promoter. To investigate h ALDH1L1 and CaMKIIa promoter activity in naive primary cultured cortical astrocytes, AAV-EF1a-DIO-mCherry virus was co-transduced. Reporter mCherry fluorescence protein can be expressed when a recombination occurs at Cre-loxP recombination sites (DIO: Double-floxed inverse Orientation) by Cre recombinase activity. AAV-CaMKIIa-Cre virus, whose promoter is known as excitatory neuron-specific, was used as a negative control. (B) Immunocytochemistry results showing mCherry expression by Cre recombinase in the condition of treatment with two viruses, AAV-hALDH1L1-Cre and AAV-EF1a-DIO-mCherry viruses. (C) Immunocytochemistry results showing no mCherry expression in negative control condition of treatment with two viruses, AAV-CaMKIIa-Cre and AAV-EF1a-DIO-mCherry viruses. Each color represents as following description. Blue indicates nuclear staining with DAPI; Green indicates astrocyte staining with Aldh1L1; Red indicates recombinase-dependent mCherry expression.

Article Snippet: AAV-EF1a-DIO-mCherry viral vector was purchased (Addgene #47636).

Techniques: Cell Culture, Expressing, Activity Assay, Fluorescence, Negative Control, Immunocytochemistry, Staining

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Scheme of the HPA axis. b, Time-line for leptin injection. c, Corticosterone levels were elevated in mice fasted for 24 hours compared to fed mice (n = 8 per group. Two-tailed unpaired t-test- ***P = 0.0006). d, I.P. administration of leptin reduced corticosterone levels in fasted mice (n = 8 vehicle, n = 9 leptin. Two-tailed unpaired t-test- *P = 0.0130). e, Schematic of optogenetic stimulation of AgRP neurons. f, One hour food intake by Agrp-IRES-Cre mice expressing ChR2 or GFP in the arcuate (n = 4 AgRPGFP, n = 6 AgRPChR2. Two-way ANOVA; Sidak’s multiple comparisons test. AgRPGFP ON vs. AgRPChR2 ON- ***P = 0.0008; AgRPChR2 OFF vs. AgRPChR2 ON- ***P = 0.0001). g, Schematic of Cre-dependent AAV-hM4Di-mCherry stereotaxic injection into the PVH of Mc4r-t2A-Cre mice. h, Three hour food intake by Mc4r-t2A-Cre mice expressing hM4Di or mCherry in the PVH (n = 9 MC4RmCherry, n = 6 MC4RhM4Di. Two-way ANOVA; Sidak’s multiple comparisons test. MC4RmCherry CNO vs. MC4RhM4Di CNO- ****P = <0.0001; MC4RhM4Di Vehicle vs. MC4RhM4Di CNO- **P = 0.0015). i, Time-line for chemogenetic inhibition of PVHMc4r neurons. j, Inhibition of PVHMc4r neurons did not affect corticosterone in fed mice (n = 9 MC4RmCherry, n = 6 MC4RhM4Di. Two-tailed unpaired t-test- P = 0.5933). k, Schematic of Cre-dependent AAV-hM3Gq-mCherry stereotaxic injection into arcuate nucleus of Pomc-IRES-Cre mice. l, Time-line for chemogenetic activation of POMC neurons. m, Activation of POMC neurons did not affect corticosterone in fasted mice (n = 9 POMCmCherry, n = 7 POMChM3Gq. Two-tailed unpaired t-test- P = 0.6736). Scale bar = 200 μm. Data represent = mean ± sem.

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Activation Assay, Injection, Two Tailed Test, Expressing, Inhibition

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematic of Cre-dependent AAV-hM3Dq-mcherry or hM4Di-mCherry injection into the arcuate of Agrp-IRES-Cre mice. b, Time-line for chemogenetic activation of AgRP neurons. c, AgRP neuron stimulation increased corticosterone in fed mice (n = 4 AgRPmCherry, n = 6 AgRPhM3Dq. Two-tailed unpaired t-test- *P = 0.0309). d, Time-line for chemogenetic inhibition of AgRP neurons. e, AgRP neuron inhibition reduced fasting corticosterone (n = 7 AgrpmCherry, n = 8 AgrphM4Di. Two-tailed unpaired t-test- **P = 0.0076). f, Schematic of photometry from PVHCrh neurons and activation of AgRP neurons. g, Representative images of GCaMP6s expression in PVHCrh neurons and hM3Dq-mCherry expression in AgRP neuron somas and terminals. h, Chemogenetic activation of AgRP neurons increased PVHCrh neuron activity (n = 4 mice). i, Mean Ca2+ signal from PVHCrh neurons after injection of CNO vs. vehicle (n = 4 mice. Two-tailed paired t-test- **P = 0.0012). j, Injection of vehicle or CNO did not affect Ca2+ signal in PVHCrh neurons in mCherry-expressing mice (n = 5 mice). k, Mean Ca2+ signal from PVHCrh neurons after injection of CNO vs. vehicle (n = 5 mice. Two-tailed paired t-test- P = 0.5297). l, Chemogenetic inhibition of AgRP neurons reduced PVHCrh neuron activity (n = 5 mice). m, Mean Ca2+ signal from PVHCrh neurons after injection of CNO vs. vehicle (n = 5 mice. Two-tailed paired t-test- **P = 0.0071). n, Schematic of optogenetic stimulation of AgRP neurons. o, Time-line for optogenetic stimulation of AgRP neurons. p, q, Optogenetic stimulation of AgRP neurons increased ACTH (n = 4 AgRPGFP, n = 5 AgRPChR2. Two-tailed unpaired t-test- **P = 0.0068) (p) and corticosterone (n = 4 AgRPGFP, n = 6 AgRPChR2, two-tailed unpaired t-test- **P = 0.0198) (q). Scale bar = 200 μm. Data represent = mean ± sem.

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Injection, Activation Assay, Two Tailed Test, Inhibition, Expressing, Activity Assay

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 1a-​-c.c. Each animal is represented by a different colour. b, Representative image of hM3Dq-mCherry expression in AgRP neuron somas in the arcuate nucleus. c, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 1a, ​,dd-​-e.e. d, Representative image of hM4Di-mCherry expression in AgRP neuron somas in the arcuate nucleus. Each animal is represented by a different colour. e, Schematics representing AAV spread (shaded regions) and fiber placement (rectangles) for every animal related to experiments in Fig. 1f-​-i.i. Each animal is represented by a different colour. See Fig. 1g for a representative histological image. f, Schematics representing AAV spread (shaded regions) and fiber placement (rectangles) for every animal related to experiments in Fig. 1f, ​,ll-​-m.m. g, Representative images of GCaMP6s expression in PVHCrh neurons, optic fiber placement in the PVH and hM4Di-mCherry expression in AgRP neuron somas in the arcuate nucleus. Each animal is represented by a different colour. h, Schematics representing fiber placements (rectangles) for every animal related to experiments in Fig. 1n-​-qq and Extended Data Fig. 1e, ​,f.f. Each animal is represented by a different colour. i, Representative image of ChR2-eYFP expression in AgRP neuron somas in the arcuate nucleus and fiber placement. Scale bar = 200 μm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 5c-​-e.e. Each animal is represented by a different colour. b, Representative image of hM4Di-mCherry expression in BNSTvGAT neuron somas. c, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Fig. 5c-​-e.e. Each animal is represented by a different colour. d, Representative image of hM4Di-mCherry expression in LHvGAT neuron somas. e, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 5f-​-i.i. Each animal is represented by a different colour. See Fig. 5g for a representative histological image. Scale bar = 200 μm. a.c. = anterior commisure, i.c. = internal capsule. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a-​-c.c. Each animal is represented by a different colour. b, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the BNST. c, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a-​-c.c. Each animal is represented by a different colour. d, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the PVH. Scale bar = 200 μm. 3V = Third ventricle. a.c. = anterior commisure. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a-​-c.c. Each animal is represented by a different colour. b, Representative image of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the LH. Scale bar = 500 μm. c, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 2a-​-c.c. Each animal is represented by a different colour. d, Representative images of ChR2-mCherry expression in AgRP neuron somas in the arcuate nucleus and fiber placement in the PBN. Left scale bar = 200 μm, Right scale bar = 500 μm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 5j-​-nn and Extended Data Fig. 2k, ​,l.l. Each animal is represented by a different colour. See Fig. 5k for a representative histological image. b, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Extended Data Fig. 1g-​-j.j. Each animal is represented by a different colour. c, Representative image of hM4Di-mCherry expression in PVHMc4r neuron somas. d, Schematics representing AAV spread (shaded regions) for every animal related to experiments in Extended Data Fig. 1k-​-m.m. Each animal is represented by a different colour. e, Representative image of hM3Dq-mCherry expression in POMC neuron somas in the arcuate nucleus. Scale bar = 200 μm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematic of collateral mapping via injection of retro-AAV-FlpO into Agrp-IRES-Cre::FLTG mice. b, AgRP neuron terminals detected upon injection of retro-AAV-FlpO into individual AgRP neuron efferent sites (denoted by row headings) in Agrp-IRES-Cre::FLTG mice. a.c: anterior commissure, 3V: third ventricle, a.q: aqueduct, s.c.p: superior cerebellar peduncle. c, Schematic of rabies-based collateral mapping. EnvA pseudotyped rabies-ΔG-GFP was injected into AgRP neuron terminal areas (PVH example) in Agrp-IRES-Cre mice that were previously injected with AAV-FLEX-TVA-mCherry in the arcuate. d, AgRP neuron terminals detected upon injection of EnvA pseudotyped rabies-ΔG-GFP into individual AgRP neuron efferent sites (denoted by row headings) in Agrp-IRES-Cre mice. e, Schematic of monosynaptic rabies mapping from PVHCrh neurons. f-j, Representative images of starter cells and putative afferents containing rabies-GFP labeled neurons in the PVH (f), BNST and mPOA (g), periventricular hypothalamus (h), LH and anterior arcuate nucleus (i) and DMH and posterior arcuate nucleus (j). PVH: paraventricular hypothalamus, a.c: anterior commissure, BNST: bed nucleus of the stria terminalis, mPOA: medial preoptic area, 3V: Third ventricle, peri VH: periventricular hypothalamus, LH: lateral hypothalamus, Arc: arcuate nucleus, DMH: dorsomedial hypothalamus. k, Schematic of recordings of BNST→PVH terminals. These mice are those used in Fig. 5j-​-o.o. l, BNST→PVH axons reduced activity in response to a looming hand motion stimulus over the animal (n = 3 mice). m, Expression of NPYRs in major PVH neuron cell types derived from single-cell RNA sequencing of PVH neurons43, plotted as average normalized expression level and fraction of cells in each cluster that express the receptor. Scale bar = 200 μm. Data represent = mean ± sem.

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Injection, Labeling, Activity Assay, Expressing, Derivative Assay, RNA Sequencing Assay

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematics representing AAV spread (shaded regions) and fiber placements (rectangles) for every animal related to experiments in Fig. 4a-​-c.c. Each animal is represented by a different colour. b-d. Representative images of ChR2-mCherry expression in AgRP neuron somas and fiber placement in the arcuate nucleus. Scale bar = 200 μm. 3V = Third ventricle. The schematics were created using The Mouse Brain in Stereotaxic Coordinates Second Edition (Paxinos and Franklin).

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Expressing

Journal: Nature

Article Title: Neural basis for fasting activation of hypothalamic-pituitary-adrenal axis

doi: 10.1038/s41586-023-06358-0

Figure Lengend Snippet: a, Schematic for BNSTvGAT→PVH neuron CRACM (n = 18 PVHCrh+ neurons, n = 9 PVHCrh− neurons/2 mice). b, Representative traces from BNSTvGAT→PVHCrh+ (upper) and BNSTvGAT→PVHCrh− (lower) recordings. c, Schematic of BNST or LH injection of Cre-dependent AAV-hM4Di-mCherry into vGAT-IRES-Cre mice. d, Time-line of chemogenetic inhibition of BNSTvGAT or LHvGAT neurons. e, Inhibition of BNSTvGat neurons increased corticosterone (BNST: n = 4 vGATmCherry, n = 4 vGAThM4Di. Two-tailed unpaired t-test- *P = 0.0131; LH: n = 5 vGATmCherry, n = 7 vGAThM4Di. Two-tailed unpaired t-test- P = 0.2837). f, Schematic of BNSTvGAT→PVH terminal inhibition. g, Representative eOpn3 expression in the BNST (upper) and location of optic fibers in the PVH (lower). h, Time-line for terminal inhibition of BNSTvGAT→PVH projections. i, Inhibition of BNSTvGAT→PVH projections increased corticosterone (n = 3 vGATmCherry, n = 5 vGATeOpn3. Two-tailed unpaired t-test- *P = 0.0076). j, Schematic of BNST→PVH terminal recordings with optogenetic stimulation of AgRP→PVH terminals. k, Representative images of synaptophysin-GCaMP6f expression in BNST neurons and terminals and ChrimsonR-tdTomato expression in AgRP neuron terminals. l, Heatmaps of BNST→PVH fiber photometry recordings from two mice. m, n, Optogenetic stimulation of AgRP→PVH terminals for 10 sec at 20 Hz (m) (n = 3 mice) and 10 min at 20 Hz (n) (n = 4 mice) reduced activity of BNST→PVH terminals. o, Representative images of BFP-labelled PVHCrh neurons, synaptophysin-GCaMP6f-expressing BNST neuron terminals and AgRP immunofluorescence (* = PVHCrh neurons apposed by BNST and AgRP neuron terminals). p, Schematic of AgRP neuron action in the PVH. Left = presynaptic disinhibition of PVHCrh neurons by GABA/presynaptic GABA-B-Rs. Right = monosynaptic inhibition of PVHMc4r neurons by GABA/postsynaptic GABA-A-Rs. Scale bar (g, k) = 200 μm. Scale bar (o) = 10 μm. Data represent = mean ± sem.

Article Snippet: For optogenetics experiments, AAV8-hSyn-DIO-mCherry (UNC Vector Core; 2.1 × 10 13 viral genomes ml −1 ), AAV9-EF1a-DIO-ChR2(H134R)-mCherry (Addgene 20297-AAV9; 2.5 × 10 13 viral genomes ml −1 ), AAV1-hSyn2-SIO-eOPN3-mScarlet-WPRE (Addgene 125713-AAV1; 1 × 10 13 viral genomes ml −1 ) or AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene 62723-AAV5; 2.1 × 10 13 viral genomes ml −1 ) was injected bilaterally into the arcuate of Agrp -IRES-Cre mice, Agrp -IRES-Cre::NPY-KO mice, Agrp -IRES-Cre::vGAT lox/lox or AgRP -IRES-Cre::vGAT lox/lox ::NPY-KO mice or the BNST of vGat-IRES-Cre mice.

Techniques: Injection, Inhibition, Two Tailed Test, Expressing, Activity Assay, Immunofluorescence

Journal: Cell reports

Article Title: Motion Streak Neurons in the Mouse Visual Cortex

doi: 10.1016/j.celrep.2020.108617

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AAV1-GCaMP6f viral vector AAV1-Syn-GCaMP6f viral vector (RRID: Addgene_100837; pAAV.Syn.GCaMP6f.WPRE.SV40, 1:1 in PBS, titer 2×10 13 ) was injected using a Nanoject II (Drummond Scientific) fitted with a glass pipette into several sites in each visual area.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Control

Journal: Cell reports

Article Title: Distinct subpopulations of ventral pallidal cholinergic projection neurons encode valence of olfactory stimuli

doi: 10.1016/j.celrep.2024.114009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A Hamilton syringe was slowly lowered to the VP and mice were injected with ~0.3 μL of the following viral vectors dependent on experiment: AAV 9 .DIO.TRE.hM4Di.P2A.m.Cherry (ADCD; packaged at UNC viral vector core), AAV 9 .RAM.d2tTA.TRE.mCherry.NLS-FLAG (RAM; Addgene # 63931, packaged at UNC viral vector core), AAV 9 .Syn.Flex-GCaMP6f.WPRE.SV40 (GCaMP6f; Addgene # 100837), AAV 8 .hSyn.DIO.hM4Di (Gi).mCherry (hM4Di; Addgene # 44362), AAV 9 .eSyn.eGFP (Vector Biolabs # VB4870), AAV 9 .RAM.d2tTA.TRE.Flex.tdTomato (FLEX-RAM; Addgene # 84468, packaged at NINDS Viral Vector Core).

Techniques: Virus, Plasmid Preparation, Recombinant, Software