Aav2 hsyn dio hm3d gq mcherry

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AAV2-hSyn-DIO-hM3D(Gq)-mCherry is a viral vector construct containing an adeno-associated virus serotype 2 (AAV2) backbone, the human synapsin promoter (hSyn), a double-inverted open reading frame (DIO) switch, the human muscarinic receptor M3 (Gq-coupled) gene (hM3D(Gq)), and the mCherry fluorescent protein gene. This vector can be used for Cre-dependent expression of the hM3D(Gq) receptor, which can be activated by the synthetic ligand clozapine-N-oxide (CNO) to induce neuronal excitation.

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8 protocols using aav2 hsyn dio hm3d gq mcherry

Recombinant AAV Viral Vectors Toolkit

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rAAV vectors:
AAV2-hSyn-hM3D(Gq)-mCherry (Addgene, LOT #v6236, CAT#50474-AAV2, 4.8×10¹³ GC/ml); AAV2-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, LOT #v58216, CAT#44361-AAV2, 2.0×10¹³ GC/ml);
AAV2-hSyn-hM4D(Gi)-mCherry (Addgene, LOT#v54503, CAT#50475-AAV2, 2.1×10¹³ GC/ml); AAV2-Syn-FLEX-ultrasensitive protein calcium sensors (GCaMP6s)-WPRE-SV40 (Vigene, 1.35X10¹³ GC/ml, Addgene plasmid #100845 packaged);
AAV5-hSyn-DIO-hM4D(Gi)-mCherry (Addgene, LOT#v47232, CAT# 44362-AAV5, 1.3×10¹³ GC/ml);
AAV5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, LOT#v54404, CAT# 44361-AAV5, 1.3×10¹³ GC/ml);
AAV2-hSyn-mCherry (Addgene, LOT #v53550, CAT#114472-AAV2, 2.6×10¹³ GC/ml);
AAV2-hSyn-DIO-mCherry (Addgene, LOT #v54505, CAT#50459-AAV2, 1.8×10¹³ GC/ml);
AAV9-hSyn-DA4.2 (Vigene Biosciences, LOT #2018.07.02, CAT#hD01, 5.45×10¹³ GC/ml); and AAV9-hSyn-DA1h (Vigene Biosciences, LOT #2019.10.18, CAT#YL10010-AAV9, 2.04×10¹³ GC/ml).
Syn, human Synapsin 1 promoter. DIO, Double-floxed inverted orientation. hM4, human M4 muscarinic receptor. hM3, human M3 muscarinic receptor. GRABDA1h (GRAB^DA), GPCR activation-based DA sensor.

Low A.Y., Goldstein N., Gaunt J.R., Huang K.P., Zainolabidin N., Yip A.K., Carty J.R., Choi J.Y., Miller A.M., Ho H.S., Lenherr C., Baltar N., Azim E., Sessions O.M., Hean Ch’ng T., Bruce A.S., Martin L.E., Halko M.A., Brady RO J.r., Holsen L.M., Alhadeff A.L., Chen A.I, & Betley J.N. (2021). Reverse-translational identification of a cerebellar satiation network. Nature.

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Targeted Optogenetic Manipulation of Retinal Cells

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Opn4^Cre/+ mice between P30-P40 were anesthetized by intraperitoneal injection of 2,2,2-Tribomoethanol. A hole was then punctured through the ora serrata using a 30 gauge needle. Each eye was then injected with 1 μL of AAV2/hSyn-DIO-hM3D(Gq)-mCherry (Addgene, viral prep #44361-AAV2) using a custom Hamilton syringe with a 33 gauge needle (Borghuis Instruments). AAVs were diluted to a titer of ~4 × 10¹⁰ viral particles/mL using filtered PBS to achieve sparse labeling. Retinas for these experiments were dissected 1–3 weeks post infection.

Sonoda T., Okabe Y, & Schmidt T.M. (2019). Overlapping morphological and functional properties between M4 and M5 intrinsically photosensitive retinal ganglion cells. The Journal of comparative neurology, 528(6), 1028-1040.

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DREADD Activation of GCG Neurons in Rats

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Introduction of DREADD: AAV2-hSyn-DIO-hM3D(Gq)-mCherry (Addgene 44361) was delivered bilaterally into the cNTS (0.5 μl; 0.1 μl/min) through a cannula implanted with the tip positioned at the following coordinates: at the level of the occipital suture, ±0.7 mm from midline; 5.9 mm ventral from skull of adult male and female Gcg-Cre Het rats (Hets: N = 10 males, 10 females; WT controls: N = 10 males, 7 females). The injector extended 2.0 mm beyond the cannula tip. Correct AAV targeting of the cNTS was confirmed at the termination of the behavioral experiments, where cannula tracks and Cre-mediated AAV reporter expression was confirmed using a fluorescence microscope.
Food-motivated behavior measurement: Rats were unilaterally injected through the cNTS cannula with CNO [0.5 μg in 0.3 μl of 1% DMSO in an artificial cerebrospinal fluid (ACSF) vehicle] or vehicle alone. Prior to the motivated behavior test, rats received 50% of their normal overnight food ration to increase the baseline motivated behavior against which the Gcg neuron activation effect was tested. Rats were placed into the testing chambers 10 min after CNO injections.

Lopez-Ferreras L., Asker M., Krieger J.P, & Skibicka K.P. (2023). Sex-divergent effects of hindbrain GLP-1-producing neuron activation in rats. Frontiers in Neuroscience, 17, 1265080.

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Targeted Viral Manipulations in Mouse VMN

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Adult mice (8 to 12 wk old) were anesthetized with isoflurane and placed in a stereotaxic apparatus. For VMN-specific deletion of the Prlr gene, groups of Prlr^lox/lox mice (n = 8/group) and a wild-type C57BL/6J group (n = 6) received bilateral 0.5-μL injections of AAV/DJ-CMV-mCherry-iCre (titer 1.4 × 10¹³ genomic copies/mL; Vector Biosystems Inc.) or control AAV/DJ-CMV-mCherry (titer 3.7 × 10¹³ genomic copies/mL; Vector Biosystems Inc.) into the VMN. For chemogenetic experiments, Prlr-iCre female mice received bilateral 0.5-μL injections of AAV2-hSyn-DIO-hM3D(Gq)-mCherry (titer 1.4 × 10¹³ genomic copies/mL, Addgene No. 44361; n = 10) or control 0.5 μL AAV5-hSyn-DIO-mCherry (titer 1.4 × 10¹³ genomic copies/mL; Addgene No. 50459; n = 7) into the VMN. For tract tracing experiments, Prlr-iCre/tdtomato females received unilateral 0.3-μL injections of AAV5-EF1a-DIO-hChR2(H13R)-eYFP-WPRE (titer 1.0 × 10¹³ genomic copies/mL; Addgene No. 20298; n = 3) into the VMN. All injections were given at a rate of 100 nL/min, and syringes were left in situ for 3 min before and 5 min after injections. Coordinates for the VMN were 1.1 mm posterior to Bregma, 0.6 mm lateral to midline, and 5.4 mm deep.

Georgescu T., Khant Aung Z., Grattan D.R, & Brown R.S. (2022). Prolactin-mediated restraint of maternal aggression in lactation. Proceedings of the National Academy of Sciences of the United States of America, 119(6), e2116972119.

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Viral Injections in VIP+ Mice

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Viral injections were performed on male and female mice (49–157 days old). Experiments were primarily performed in VIP^+/cre individuals (Figs. 6 and 7 and Supplementary Figs. 8 and 9). For additional validation of viral specificity (Supplementary Fig. 7b, c), injections were performed in VIP^+/+ animals. For electrophysiological validation of the Gi-DREADD approach (Supplementary Fig. 7d–f) mice were heterozygous for both VIP-IRES-Cre and Ai32 (i.e. VIP^+/cre; Ai32^+/−).
Mice were anaesthetised using 1% isoflurane in O2, and placed into a stereotaxic frame following which the skull was exposed. Craniotomy and bilateral injection of virus was completed using a motorised injector under computer control (Drill and 473 Microinjection Robot: Neurostar, Tϋbingen, Germany). Subsequently, 69 nl of viral vector encoding an activating or inhibiting DREADD or control construct (AAV2-hSyn-DIO-hM3D(Gq)-mCherry, AAV2-hSyn-DIO-hM4D(Gi)-mCherry or AAV2-hSyn-DIO-mCherry respectively; Addgene, MA, USA) was delivered at 25 nl/s. Micropipettes were left in place for 5 min following injection, and were then retracted in 100 µm/5s increments until above the SCN.

Paul S., Hanna L., Harding C., Hayter E.A., Walmsley L., Bechtold D.A, & Brown T.M. (2020). Output from VIP cells of the mammalian central clock regulates daily physiological rhythms. Nature Communications, 11, 1453.

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Diverse AAV-Based Neurogenetic Toolkits

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AAV2/5-Ef1a-DIO hChR2(E123T/T159C)-EYFP, AAV1-hSyn-Cre, AAV9-CAG-FLEX-GCaMP6s-WPRE-SV40 and AAV9-hSyn-eGFP were obtained from the Vector Core of the University of Pennsylvania. AAV9-CAG-FLEX-tdTomato, AAV2-mCherry-FLEX-dtA, AAV9-hSyn-DIO-mCherry-2A-SybGFP, AAV2-hSyn-DIO-hM4Di-mCherry and AAV2-Syn-DIO-GFP were produced by the Vector Core of the University of North Carolina. AAVDJ-CAG-FLEX-TVA-mCherry was obtained from viral vector core at Salk Institute. AAVretro-CAG-GFP (Addgene # 37825-AAVrg), AAV1-CAG-FLEX-jGCaMP7s-WPRE (Addgene#104495-AAV1), AAV5-hSyn-FLEX-ChrimsonR-tdTomato (Addgene # 62723) and AAV2-hSyn-DIO-hM3D(Gq)-mCherry (Addgene #44361-AAV2) were produced by Addgene. AAV2(retro)-CAG-iCre (Addgene# 81070) was produced by Vector Biolabs. AAV2/9-hSyn-FLEX-TVA-P2A-EGFP-2A-oG and EnvA-SAD-ΔG-mCherry were gifts from Dr. Yuanyuan Liu at NIDCR/NIH. AAV8-CAG-FLEX-TCB (TVA-mCherry), AAV-EF1a-DIO-HB and EnvA-SAD-ΔG-GFP were obtained from GT3 Core Facility of the Salk Institute. All viral vectors were stored in aliquots at −80°C until use.

Gu X., Zhang Y.Z., O’Malley J.J., De Preter C.C., Penzo M, & Hoon M.A. (2023). Neurons in the caudal ventral lateral medulla mediate descending pain control. Nature neuroscience, 26(4), 594-605.

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Viral Vector Toolkit for Neuroscience

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AAV2/5-Ef1a-DIO hChR2(E123T/T159C)-EYFP and AAV9-hSyn-FLEX-GCaMP6s-WPRE-SV40 were produced by the Vector Core of the University of Pennsylvania. AAV9-EF1a-DIO-eNpHR3.0-mCherry, AA9-CAG-FLEX-tdTomato, AV9-hSyn-DIO-mCherry-2A-SyneGFP and AAV2-Syn-DIO-GFP were produced by the Vector Core of the University of North Carolina. AAV5-Syn-FLEX-ChrimsonR-tdTomato (Addgene plasmid # 62723) and AAV2-hSyn-DIO-hM4Di-mCherry, AAV2-hSyn-DIO-hM3D(Gq)-mCherry were produced by Addgene. AAV2(retro)-CAG-iCre (Addgene plasmid # 81070) was produced by Vector Biolabs. AAV9-EF1a-FLEX-TVA-mCherry (Addgene plasmid # 38044) and AAV9-CAG-FLEX-RG (Addgene plasmid # 38043) were produced by Vigene Biosciences, Inc. EnvA-SAD-ΔG-eGFP (Addgene plasmid # 32635) was produced by the Viral Vector Core of the Salk Institute for Biological Studies. AAV2-retro-CAG-tdTomato-WPRE is a gift from Dr. Yuanyuan Liu at NIDCR. All viral vectors were stored in aliquots at −80°C until use.

Sofia Beas B., Gu X., Leng Y., Koita O., Rodriguez-Gonzalez S., Kindel M., Matikainen-Ankney B.A., Larsen R.S., Kravitz A.V., Hoon M.A, & Penzo M.A. (2020). A ventrolateral medulla-midline thalamic circuit for hypoglycemic feeding. Nature Communications, 11, 6218.

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Chemogenetic Manipulation of DMH Neurons

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Arg3.1-Cre/ER T2 mice (12-20-week-old) were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), and positioned on a stereotaxic instrument (Narishige, Tokyo, Japan). Mice were injected in each side or one side of the DMH with ~0.5 µL AAV2-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, Cambridge, USA), AAV2-hSyn-DIO-hM4D(Gi)-mCherry (Addgene), AAV8-eSyn-DIO-EGFP (Addgene) or AAV8-eSyn-DIO-eNpHR3.0-EGFP (Vector Biolabs, Malvern, PA, USA) using the following coordinates: AP: -1.85 mm, L:  0.3 mm, DV: -5.5 mm. Open wounds were sutured after viral injection. A 7-14-day recovery period was allowed before experiments were started. Mice were fasted overnight (16 h) and received 4-OHT (10 mg/kg) injection (i.p.) 1 h after refeeding to induce Cre recombination. To ensure adequate expression of proteins, experiments were carried out at least 7 days after the 4-OHT injection.
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Imoto D., Yamamoto I., Matsunaga H., Yonekura T., Lee M., Kato K., Yamasaki T., Otsuguro K., Horiuchi M., Iijima N., Kimura K., & Toda C. (2020). Activation of prodynorphin neurons in the dorsomedial hypothalamus inhibits food intake and promotes positive valence.

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