Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 3. vlPAG activity is required for systemic morphine antinociception and drives spinal NA–dependent antinociception. (A) Bilateral vlPAG AAV-DIO-hM4Di- mCherry injections in Vglut2-cre mice with representative image of viral expression. Scale bar, 500 μm. (B) Left: Hot plate withdrawal latencies of vlPAGVglut2-cre::hM4Di mice administered CNO (3 mg/kg i.p.) versus saline without (light green bars) and with morphine (5 mg/kg s.c.) (dark green bars) (two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 13 mice; saline versus CNO effect, P < 0.0001, F1,12 = 56.93). Right: Withdrawal latencies of non–virus-injected controls (two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 9 mice; saline versus CNO effect, P = 0.32, F1,8 = 1.143). (C) Bilateral vlPAG AAV-DIO-hM3Dq- mCherry injections in Vglut2-cre mice, combinations of systemic CNO with intrathecal antagonists, and representative image of viral expression. Scale bar, 500 μm. (D) Left: Withdrawal latencies after systemic saline or CNO and intrathecal saline, phentolamine (5 μg), or naltrexone (5 μg) (Friedman test with Dunn’s multiple comparisons test, n = 12 subjects, P < 0.0001, Friedman statistic = 26.40). Right: Von Frey mechanical thresholds (Friedman test with Dunn’s multiple comparisons test, n = 12 subjects, P = 0.0001, Friedman statistic = 21.00). (E) Representative images of c-Fos immunohistochemistry in TH-positive LC neurons of vlPAGVglut2-cre::hM3Dq mice after systemic injection of saline (top) or CNO (bottom). Scale bars, 150 μm. Right: % of TH-positive LC neurons that colocalize with green c-Fos signal (five to eight images analyzed per mouse; n = 6 saline, n = 5 CNO, two-sided Mann-Whitney test). Data in each graph reported as means ± SEM.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Activity Assay, Expressing, Saline, Virus, Injection, Immunohistochemistry, MANN-WHITNEY

Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 4. Anatomical characterization of inputs to the LC from the vlPAG. (A) vlPAG injection of AAV-DIO-tdTom in Vglut2- or Vgat-cre mice. (B) Vglut2-cre injection site. (C) vlPAGVglut2-cre terminals (magenta) and TH (green) in the peri-LC. Bar, Barrington’s nucleus. (D) Vgat-cre injection site. (E) vlPAGVgat-cre terminals. Scale bars, 300 μm [(B) to (E)]. (F) Quantification of magenta and green pixel intensity in the peri-LC of vlPAGVglut2-cre::tdTom mice normalized by z score (n = 6 LC slices from three mice). (G) Same as (F) for vlPAGVgat-cre::tdTom. (H) Left: Injections of AAVretro-cre in the LC and AAV-DIO-mCherry in the vlPAG of wild-type mice. Middle: Image of mCherry+ vlPAG neurons. Right: Resulting terminals in the LC and RVM. Scale bars, 500 μm. (I) Same as (H) for vlPAG→RVM neurons. Scale bars, 500 μm. (J) Left: Orthogonal recombinase strategy to label vlPAG neurons that project to the RVM and LC. Middle: Representative image of mCherry (magenta) and YFP (green). Arrows indicate double-labeled (white) neu- rons. Scale bar, 300 μm. Right: Quantification of mCherry and YFP in the vlPAG.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Injection, Labeling

Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 8. Pathway-specific modulation of vlPAG and RVM terminals in the LC modulates nociceptive behavior. (A) Left: Bilateral cannula placement over the LC in uninjected control mice. Right: Hot plate withdrawal latencies of control mice microinfused in the LC with saline (150 nl) versus CNO (3 μM, 150 nl) without (light gray) and with morphine (5 mg/kg s.c.) (dark gray; two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 8 mice; saline versus CNO effect, P = 0.4178, F1,7 = 0.7412). (B) Left: Bilateral viral injection of AAV-DIO-hM4Di-mCherry in the vlPAG of Vglut2-cre mice with bilateral cannula placement over the LC. Right: Hot plate withdrawal latencies after microinfusion with saline versus CNO without (light green) and with morphine (5 mg/kg s.c.) (dark green; two-way repeated-measures ANOVA with Sidak’s multiple comparisons test; n = 12 mice; saline versus CNO effect, P = 0.0022, F1,11 = 15.72). (C) Left: Bilateral viral injection of AAV-DIO-hM4Di-mCherry in the RVM of Vgat-cre mice with bilateral cannula placement over the LC. Right: Hot plate withdrawal latencies after microinfusion of saline versus CNO (blue bars, n = 12 mice), microinfusion of CNO with intrathecal injections of saline versus phentolamine (5 μg, light blue bars, n = 9 mice), and microinfusion of saline versus CNO with morphine (5 mg/kg s.c.) (dark blue, n = 12 mice; mixed-effects analysis with matching across row and Tukey’s multiple comparisons test, P < 0.0001, F2.560,25.09 = 27.36). (D) Circuit diagram of DPMS inputs to the LC and their opioid sensitivity. Data in each graph reported as means ± SEM.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Control, Saline, Injection

Journal: Cell

Article Title: A Genetically Defined Compartmentalized Striatal Direct Pathway for Negative Reinforcement

doi: 10.1016/j.cell.2020.08.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-MOR Immunostar 24216 Rabbit polyclonal anti-tyrosine hydroxylase Millipore AB152 Chicken polyclonal anti-GFP Aves Labs GFP1020 Rabbit polyclonal anti-RFP Rockland 600-401-379 Rabbit monoclonal anti-HA-Tag Cell Signaling 3724S Mouse monoclonal anti-Parvalbumin Millipore MAB1572 Rabbit polyclonal anti-Somatostatin-14 Peninsula Laboratories T-4103 Goat polyclonal anti-ChAT Millipore AB144P Bacterial and Virus Strains AAV8-Ef1a-fDIO-GCaMP6m Laboratory of Karl Deisseroth N/A AAVdj-hSyn-Cre OFF /Flp ON -hChR2(H134R)-eYFP Fenno et al., 2014 Addgene 55648 AAVdj-hSyn-Cre ON /Flp OFF -hChR2(H134R)-eYFP Fenno et al., 2014 Addgene 55646 AAV8-EF1a-fDIO-Cre-p2A-mCherry Laboratory of Karl Deisseroth N/A AAV5-Ef1a-DIO-hChR2(H134R)-eYFP UNC N/A AAVdj-EF1a-fDIO-eYFP UNC N/A AAV8-hSyn-DIO-KORD-IRES-Mcitrine UNC N/A AAV2/8-Ef1a-fDIO-TVA-mCherry Laboratory of Z. Josh Huang N/A retroAAV2-CBA-fDIO-Cre Vigene Biosciences N/A AAV9-CAGGS-Flex-mKate-T2A-TVA HHMI Janelia Research Campus N/A AAV9-CAGGS-Flex-mKate-T2A-N2c-G HHMI Janelia Research Campus N/A Rbv-CVS-N2c-dG-GFP HHMI Janelia Research Campus Addgene 73461 AAV8-hSyn-DIO-mCherry Addgene Addgene 50459 Experimental Models: Organisms/Strains Mouse: Tg(Drd1a-cre)FK150Gsat/Mmucd (the “D1-Cre” line) MMRRC RRID: MMRRC_029178-UCD Mouse: B6.FVB(Cg)-Tg(Adora2a-cre) KG139Gsat/Mmucd (the “A2A-Cre” line) MMRRC RRID: MMRRC_036158-UCD Mouse: Tshz1-2A-FlpO This study N/A Mouse: Frt-Stop-Frt-TdTomato He et al., 2016 N/A Mouse: Pdyn-IRES-Cre (B6.Cg-129S-Pdyn tm1.1(cre)Mjkr/LowlJ) The Jackson Laboratory JAX: 027958 Mouse: Ai14 (B6.Cg-Gt(ROSA) 26Sortm14(CAG-tdTomato)Hze/J) The Jackson Laboratory JAX: 007908 Software and Algorithms ImageJ (Fiji) software NIH https://fiji.sc/ MATLAB Mathworks https://www.mathworks.com/ GraphPad Prism 7 GraphPad Software https://www.graphpad.com/ Open in a separate window KEY RESOURCES TABLE Tshz1 labels a population of striatal direct pathway medium spiny neurons (dMSNs) Tshz1 -expressing ( Tshz1 + ) dMSNs are localized in the striosome Tshz1 + striosomal dMSNs represent punishment and drive negative reinforcement Pdyn labels another population of striosomal dMSNs mediating positive reinforcement

Techniques: Software

Journal: Neuron

Article Title: Loss of nigral excitation of cholinergic interneurons contributes to Parkinsonian motor impairments

doi: 10.1016/j.neuron.2021.01.028

Figure Lengend Snippet: (A) Schematic of design and injection of AAV-DIO-mGluR1-FLAG in ChAT-Cre mice.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-TH Millipore Cat# AB152 Donkey anti-rabbit Alexa Fluor 488 Life Technologies Cat# A21206 Rabbit anti-DDDDK tag Abcam Cat# ab1162 Donkey anti-rabbit Alexa Fluor 594 Abcam Cat# ab150076 Bacterial and Virus Strains AAV5.EF1a.DIO.hChR2 (H134R)-EYFP pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA was a gift from Karl Deisseroth Addgene plasmid: 20298 AAV9.hSyn.tdTomato.T2A.mGIRK2-1 Marcott et al., 2014 Penn Vector Core: V3992MI-R AAV5-hSyn-DIO-hM3D-mCherry Krashes et al., 2011 Addgene plasmid: 44361 AAV5-EF1a-mCherry-Cre UNC Vector Core AV6144B AAVDJ-DIO-mGluR1-myc-FLAG This manuscript Addgene plasmid: 166227 Chemicals, Peptides, and Recombinant Proteins Alexa Fluor 594 Thermo Fisher Cat# A10436 Cal520 ATT Bioquest Cat# 21141 6-hydroxydopamine hydrobromide Tocris Cat# 2547 Desipramine hydrochloride Tocris Cat# 3067 Pargyline hydrochloride Sigma Cat# P8013 DNQX Tocris Cat# 0189 Picrotoxin Tocris Cat# 1128 MK-801 Tocris Cat# 0924 JNJ16259685 Tocris Cat# 2333 L-aspartate Tocris Cat# 0214 Sulforhodamine 101 Sigma Cat# S7635 Sulpiride Tocris Cat# 0895 Clozapine N-oxide Tocris Cat# 4936 (RS)-3,5-DHPG Tocris Cat# 0342 Substance P Tocris Cat# 1156 Bafilomycin A1 Tocris Cat# 1334 BAPTA Acros Organic Cat# 123311000 Critical Commercial Assays PerfeCTa ToughMix Low Rox Quantabio Cat# 95112 SMART-Seq HT kit Takara Cat# 634455 Experimental Models: Cell Lines HEK-239T cells ATCC Cat# 11268 Experimental Models: Organisms/Strains Mouse / DAT-IRES-Cre Jackson Labs JAX: 006660 Mouse / ChAT-IRES-Cre Jackson Labs JAX: 006410 Mouse / Floxed VGLUT2 Jackson Labs JAX: 007583 Mouse / CaMKII-Cre Jackson Labs JAX: 005359 Mouse / C57Bl6 Jackson Labs JAX: 000664 Oligonucleotides B-actin IDT mmPT: 39a.2214843.g Chat IDT mmPT: 58.31626536 Ctip2 IDT mmPT: 58.31107142 Grm1 IDT mmPT: 58.8395588 Recombinant DNA mGluR1-Myc/DDK Origene Cat# MR211136 Software and Algorithms Axograph X Axograph Scientific RRID SCR_014284 https://axograph.com Prism GraphPad Software RRID SCR_002798 https://www.graphpad.com/scientific-software/prism Toronado Pressler & Strowbridge, 2019 https://github.com/StrowbridgeLab/Toronado-Laser-Scanning Open in a separate window KEY RESOURCES TABLE.

Techniques: Injection

Journal: Neuron

Article Title: Loss of nigral excitation of cholinergic interneurons contributes to Parkinsonian motor impairments

doi: 10.1016/j.neuron.2021.01.028

Figure Lengend Snippet: (A) Representative traces from DLS ChIs from saline (left), 6-OHDA (middle) and 6-OHDA + mGluR1 overexpressing (right) groups following photostimulation (5 flashes, 2 ms, 20 Hz) of midbrain inputs.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-TH Millipore Cat# AB152 Donkey anti-rabbit Alexa Fluor 488 Life Technologies Cat# A21206 Rabbit anti-DDDDK tag Abcam Cat# ab1162 Donkey anti-rabbit Alexa Fluor 594 Abcam Cat# ab150076 Bacterial and Virus Strains AAV5.EF1a.DIO.hChR2 (H134R)-EYFP pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA was a gift from Karl Deisseroth Addgene plasmid: 20298 AAV9.hSyn.tdTomato.T2A.mGIRK2-1 Marcott et al., 2014 Penn Vector Core: V3992MI-R AAV5-hSyn-DIO-hM3D-mCherry Krashes et al., 2011 Addgene plasmid: 44361 AAV5-EF1a-mCherry-Cre UNC Vector Core AV6144B AAVDJ-DIO-mGluR1-myc-FLAG This manuscript Addgene plasmid: 166227 Chemicals, Peptides, and Recombinant Proteins Alexa Fluor 594 Thermo Fisher Cat# A10436 Cal520 ATT Bioquest Cat# 21141 6-hydroxydopamine hydrobromide Tocris Cat# 2547 Desipramine hydrochloride Tocris Cat# 3067 Pargyline hydrochloride Sigma Cat# P8013 DNQX Tocris Cat# 0189 Picrotoxin Tocris Cat# 1128 MK-801 Tocris Cat# 0924 JNJ16259685 Tocris Cat# 2333 L-aspartate Tocris Cat# 0214 Sulforhodamine 101 Sigma Cat# S7635 Sulpiride Tocris Cat# 0895 Clozapine N-oxide Tocris Cat# 4936 (RS)-3,5-DHPG Tocris Cat# 0342 Substance P Tocris Cat# 1156 Bafilomycin A1 Tocris Cat# 1334 BAPTA Acros Organic Cat# 123311000 Critical Commercial Assays PerfeCTa ToughMix Low Rox Quantabio Cat# 95112 SMART-Seq HT kit Takara Cat# 634455 Experimental Models: Cell Lines HEK-239T cells ATCC Cat# 11268 Experimental Models: Organisms/Strains Mouse / DAT-IRES-Cre Jackson Labs JAX: 006660 Mouse / ChAT-IRES-Cre Jackson Labs JAX: 006410 Mouse / Floxed VGLUT2 Jackson Labs JAX: 007583 Mouse / CaMKII-Cre Jackson Labs JAX: 005359 Mouse / C57Bl6 Jackson Labs JAX: 000664 Oligonucleotides B-actin IDT mmPT: 39a.2214843.g Chat IDT mmPT: 58.31626536 Ctip2 IDT mmPT: 58.31107142 Grm1 IDT mmPT: 58.8395588 Recombinant DNA mGluR1-Myc/DDK Origene Cat# MR211136 Software and Algorithms Axograph X Axograph Scientific RRID SCR_014284 https://axograph.com Prism GraphPad Software RRID SCR_002798 https://www.graphpad.com/scientific-software/prism Toronado Pressler & Strowbridge, 2019 https://github.com/StrowbridgeLab/Toronado-Laser-Scanning Open in a separate window KEY RESOURCES TABLE.

Techniques:

Journal: Neuron

Article Title: Loss of nigral excitation of cholinergic interneurons contributes to Parkinsonian motor impairments

doi: 10.1016/j.neuron.2021.01.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-TH Millipore Cat# AB152 Donkey anti-rabbit Alexa Fluor 488 Life Technologies Cat# A21206 Rabbit anti-DDDDK tag Abcam Cat# ab1162 Donkey anti-rabbit Alexa Fluor 594 Abcam Cat# ab150076 Bacterial and Virus Strains AAV5.EF1a.DIO.hChR2 (H134R)-EYFP pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA was a gift from Karl Deisseroth Addgene plasmid: 20298 AAV9.hSyn.tdTomato.T2A.mGIRK2-1 Marcott et al., 2014 Penn Vector Core: V3992MI-R AAV5-hSyn-DIO-hM3D-mCherry Krashes et al., 2011 Addgene plasmid: 44361 AAV5-EF1a-mCherry-Cre UNC Vector Core AV6144B AAVDJ-DIO-mGluR1-myc-FLAG This manuscript Addgene plasmid: 166227 Chemicals, Peptides, and Recombinant Proteins Alexa Fluor 594 Thermo Fisher Cat# A10436 Cal520 ATT Bioquest Cat# 21141 6-hydroxydopamine hydrobromide Tocris Cat# 2547 Desipramine hydrochloride Tocris Cat# 3067 Pargyline hydrochloride Sigma Cat# P8013 DNQX Tocris Cat# 0189 Picrotoxin Tocris Cat# 1128 MK-801 Tocris Cat# 0924 JNJ16259685 Tocris Cat# 2333 L-aspartate Tocris Cat# 0214 Sulforhodamine 101 Sigma Cat# S7635 Sulpiride Tocris Cat# 0895 Clozapine N-oxide Tocris Cat# 4936 (RS)-3,5-DHPG Tocris Cat# 0342 Substance P Tocris Cat# 1156 Bafilomycin A1 Tocris Cat# 1334 BAPTA Acros Organic Cat# 123311000 Critical Commercial Assays PerfeCTa ToughMix Low Rox Quantabio Cat# 95112 SMART-Seq HT kit Takara Cat# 634455 Experimental Models: Cell Lines HEK-239T cells ATCC Cat# 11268 Experimental Models: Organisms/Strains Mouse / DAT-IRES-Cre Jackson Labs JAX: 006660 Mouse / ChAT-IRES-Cre Jackson Labs JAX: 006410 Mouse / Floxed VGLUT2 Jackson Labs JAX: 007583 Mouse / CaMKII-Cre Jackson Labs JAX: 005359 Mouse / C57Bl6 Jackson Labs JAX: 000664 Oligonucleotides B-actin IDT mmPT: 39a.2214843.g Chat IDT mmPT: 58.31626536 Ctip2 IDT mmPT: 58.31107142 Grm1 IDT mmPT: 58.8395588 Recombinant DNA mGluR1-Myc/DDK Origene Cat# MR211136 Software and Algorithms Axograph X Axograph Scientific RRID SCR_014284 https://axograph.com Prism GraphPad Software RRID SCR_002798 https://www.graphpad.com/scientific-software/prism Toronado Pressler & Strowbridge, 2019 https://github.com/StrowbridgeLab/Toronado-Laser-Scanning Open in a separate window KEY RESOURCES TABLE.

Techniques: Plasmid Preparation, Recombinant, Software

Journal: Cell reports

Article Title: VIP interneurons regulate cortical size tuning and visual perception

doi: 10.1016/j.celrep.2023.113088

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For imaging experiments, we injected 200nL of adenoassociated virus (AAVdj-ef1a-fDIO-GCaMP6m (plasmid gift of K. Deisseroth lab, Stanford), AAV5-Syn-GCaMP6s (Addgene # 100843), or AAV5-Syn-FLEX-GCaMP6s (Addgene # 100845); diluted to a titer of 10^12) unilaterally at three sites (in mm from bregma): AP 3.5, ML 1.5, DV 0.4; AP 3, ML 2, DV 0.4; AP 2.5, ML 2.5, DV 0.4.

Techniques: Virus, Plasmid Preparation, Software