Journal: Current biology : CB

Article Title: Precise Coordination of Three-dimensional Rotational Kinematics by Ventral Tegmental Area GABAergic Neurons

doi: 10.1016/j.cub.2019.08.022

Figure Lengend Snippet: (A) Unilateral stimulation of VTAVgat+ neurons. AAV-DIO-ChR2 or AAV-SIO-StGtACR2 was injected into the VTA of Vgat-ires-Cre mice. Inset shows ChR2 in the vicinity of TH-positive cells. Note glial scarring caused by optic fiber above VTA.

Article Snippet: rAAV5.EF1α.DIO.hChR2(H134R).eYFP and rAAV5.EF1α.DIO.eYFP were obtained from the Duke University Vector Core. pAAV-hSyn1-SIO-stGtACR2-FusionRed (Mahn et al., 2018) and pGP-AAV-syn-jGCaMP7f-WPRE (Dana et al., 2018) were obtained from Addgene. pGP-AAV-syn-jGCaMP7f-WPRE was from Douglas Kim (Addgene viral prep # 104488-AAV1; http://n2t.net/addgene:104488 ; RRID:Addgene_104488). pAAV_hSyn1-SIO-stGtACR2-FusionRed was from Ofer Yizhar (Addgene viral prep # 105677-AAV1; http://n2t.net/addgene:105677 ; RRID:Addgene_105677).

Techniques: Injection

Journal: Current biology : CB

Article Title: Precise Coordination of Three-dimensional Rotational Kinematics by Ventral Tegmental Area GABAergic Neurons

doi: 10.1016/j.cub.2019.08.022

Figure Lengend Snippet: Key Resources Table

Article Snippet: rAAV5.EF1α.DIO.hChR2(H134R).eYFP and rAAV5.EF1α.DIO.eYFP were obtained from the Duke University Vector Core. pAAV-hSyn1-SIO-stGtACR2-FusionRed (Mahn et al., 2018) and pGP-AAV-syn-jGCaMP7f-WPRE (Dana et al., 2018) were obtained from Addgene. pGP-AAV-syn-jGCaMP7f-WPRE was from Douglas Kim (Addgene viral prep # 104488-AAV1; http://n2t.net/addgene:104488 ; RRID:Addgene_104488). pAAV_hSyn1-SIO-stGtACR2-FusionRed was from Ofer Yizhar (Addgene viral prep # 105677-AAV1; http://n2t.net/addgene:105677 ; RRID:Addgene_105677).

Techniques: Virus, Plasmid Preparation, Software

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